Compositions comprising e-selectin antagonists and uses therefor

ABSTRACT

This invention discloses the use of an E-selectin antagonist and a mobilizer of hematopoietic stem cells or progenitor cells in methods and compositions for treating or preventing immunocompromised conditions resulting from medical treatment. The present invention is particular relevant for prophylaxis and/or treatment of hematopoeitic disorders including neutropenia, agranulocytosis, anemia and thrombocytopenia in individuals receiving or proposed to receive treatments that target rapidly dividing cells or that disrupt the cell cycle or cell division.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/747,324,which issued as U.S. Pat. No. 9,254,322 on Feb. 9, 2016, and which isthe U.S. National Stage Application of PCT Application No.PCT/AU08/01810, filed Dec. 9, 2008, and which claims priority to U.S.Provisional Application No. 61/012,756, filed Dec. 10, 2007, all ofwhich are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 27, 2016, isnamed “Sequence Listing.txt” and is 97,575 bytes in size.

FIELD OF THE INVENTION

This invention relates generally to the use of an E-selectin antagonistand a mobilizer of hematopoietic stem cells or progenitor cells inmethods and compositions for treating or preventing immunocompromisedconditions resulting from medical treatment. The present invention isparticular relevant for prophylaxis and/or treatment of hematopoeiticdisorders including neutropenia, agranulocytosis, anemia andthrombocytopenia in individuals receiving or proposed to receivetreatments that target rapidly dividing cells or that disrupt the cellcycle or cell division.

Bibliographic details of certain publications numerically referred to inthis specification are collected at the end of the description.

BACKGROUND OF THE INVENTION

Hematopoiesis is an essential, lifelong process whereby highlyspecialized blood cells are generated from hematopoietic stem cells,including cells responsible for carbon dioxide and oxygen transport(erythrocytes), blood clotting (platelets), humoral immunity (Blymphocytes), cellular immunity (T lymphocytes), as well as cells whichrespond to foreign organisms and their products (granulocytes,monocytes, and macrophages).

Mature functional end cells and their immediate precursors have alimited life-span and a limited proliferative capacity and hence are notself-maintaining. Thus, these cells are continuously replaced from apool of more primitive proliferating progenitor cells. The proliferationand self-renewal of these cells depend on stem cell factor (SCF).Glycoprotein growth factors regulate the proliferation and maturation ofthe cells that enter the blood from the marrow, and cause cells in oneor more committed cell lines to proliferate and mature. Three morefactors which stimulate the production of committed stem cells arecalled colony-stimulating factors (CSFs) and includegranulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF) andmacrophage CSF (M-CSF).

Under normal conditions, senescent mature cells are continuously removedand replaced with newly generated cells. Under stress conditions, theremay be an increased rate at which blood cells are destroyed or lost, orthere may be a compromised capacity to replenish cells undergoing normalsenescent attrition, resulting in depletion of erythrocytes (anemia),platelets (thrombocytopenia), leukocytes (leukopenia) includingneutrophil granulocytes (neutropenia), and/or agranulocytosis (completeabsence of white cells).

Radiation and chemotherapeutic treatment frequently produce severereversible neutropenia or agranulocytosis, thrombocytopenia, and anemia.This effect comes about as the result of the toxicity of these treatmentregimens on dividing hematopoietic stem cells and the consequentdepletion of hematopoietic precursors and of the cells responsible forproducing the required CSFs and hematopoietic potentiators. Thedepletion of hematopoietic precursors in the bone marrow associated withchemotherapy and irradiation sometimes results in life-threateninghemorrhagic and infectious complications. Severe suppression ofhematopoiesis is a major factor in limiting chemotherapy use and doseescalation. Replacement of depleted blood cell types by transfusion isnot always practical or desirable as it often affords only temporaryimprovement, is expensive, and is associated with risks of infection,fluid overload, and immune-mediated adverse reactions. Thus there hasbeen intense interest in developing methods of using hematopoietic CSFsand potentiators to treat neutropenia, agranulocytosis,thrombocytopenia, and anemia.

In recent years three recombinant human hematopoietic growth factorsbecame available for clinical use: erythropoietin (EPO) for stimulatingthe production of erythrocytes in the treatment of anemia, as well asG-CSF and GM-CSF for stimulating the production of neutrophils in thetreatment of neutropenia. Apart from their effect on stimulatinggranulopoiesis, G-CSF and GM-CSF also mobilize large numbers ofhematopoietic stem and progenitor cells (HSPCs) from the bone marrowinto the peripheral blood, which further accelerates reconstitution ofthe hematopoietic system. HSPC mobilization is mediated by severalfactors including trans-acting signals that originate from the releaseof proteases including serine- and metallo-proteinases whose substratesinclude various molecules implicated in progenitor trafficking such asVCAM-1 membrane-bound Kit ligand, the c-Kit receptor, stromal-derivedfactor-1 (SDF-1 or CXCL12) and its cognate receptor CXCR4.

In work leading up to the present invention, it was discovered thatE-selectin, a Ca²⁺-dependent adhesion molecule expressed by bone marrowendothelial sinuses and on inflamed endothelial cells, regulateshematopoietic stem cell turn-over in the bone marrow. In particular, thepresent inventors determined that the absence of E-selectin at theendothelial niche significantly delays hematopoietic stem cell turnoverand that blocking E-selectin mediated adhesive interactions protectshematopoietic stem cells from medical treatments that target rapidlydividing cells, including myeloablative therapies such as radiation andchemotherapeutic treatments. Accordingly, it was proposed thatE-selectin antagonists would be useful for treating or preventingimmunocompromised conditions such as neutropenia, agranulocytosis,thrombocytopenia, and anemia, which result from medical treatment.

SUMMARY OF THE INVENTION

The present invention is predicated in part on the determination thatmobilization of hematopoietic stem cells and progenitor cells bymobilizing agents (also referred to herein as “mobilizers” or “mobilizerof hematopoietic stem cells or progenitor cells”) such as G-CSF issignificantly enhanced by co-administration of an E-selectin antagonist.This in turn results in higher numbers of hematopoietic stem cells,progenitor cells and granulocytes such as neutrophils in peripheralblood when compared to administration of stem cell mobilizers alone.Based on this determination, the present inventors propose thatconcurrent administration of an E-selectin antagonist and a mobilizer ofhematopoietic stem cells or progenitor cells is useful in compositionsand methods for stimulating or enhancing hematopoiesis and for treatingor preventing immunocompromised conditions that result from medicaltreatment, as described hereafter.

Accordingly, in one aspect, the present invention provides compositionsfor stimulating or enhancing hematopoiesis or for treating or preventingan immunocompromised condition in a subject, which condition resultsfrom exposure of the subject to a medical treatment. These compositionsgenerally comprise an E-selectin antagonist and a mobilizer ofhematopoietic stem cells or progenitor cells. In some embodiments, themobilizer is characterized by its ability to decrease or block theexpression, synthesis or function of CXCL12 or is characterized by itsability to block or antagonize CXCR4. For example, illustrativemobilizers can be selected from: (1) small organic molecules (e.g.,AMD3100); (2) polypeptides such as but not limited to: a cytokine (e.g.,interleukin-1 (IL-1), interleukin-3 (IL-3), interleukin-6 (IL-6),interleukin-11 (IL-11), interleukin-7 (IL-7), and interleukin-12(IL12)), a colony stimulating factor (e.g., granulocyte colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), macrophage colony stimulating factor (M-CSF), stem cellfactor, FLT-3 ligand or a combination thereof), a protease (e.g.,metalloproteinase (like MMP2 or MMP9) a serine protease, (like cathepsinG, or elastase) a cysteine protease (like cathepsin K) and a dipeptidylpeptidase-1 (DDP-1 OR CD26)) and a chemokine (e.g., CXCL12, IL-8,Mip-1α, and Groβ; (3) DNA or RNA molecules (e.g., a small interferingRNA (siRNA) molecule or an antisense molecule specific for CXCL12) and(4) carbohydrates (e.g., a sulfated carbohydrate such as but not limitedto Fucoidan and sulfated dextran). In specific embodiments, themobilizer is a colony stimulating factor such as G-CSF.

Non limiting examples of suitable E-selectin antagonists include smallmolecules, such as nucleic acids, peptides, polypeptides,peptidomimetics, carbohydrates, lipids or other organic (carboncontaining) or inorganic molecules. Suitably, the E-selectin antagonistis selected from antigen-binding molecules that are immuno-interactivewith E-selectin, peptides that bind to E-selectin and that blockcell-cell adhesion, and carbohydrate or peptide mimetics of E-selectinligands. In some embodiments, the E-selectin antagonist reduces theexpression of an E-selectin gene or the level or functional activity ofan expression product of that gene. For example, the E-selectinantagonist may antagonize the function of E-selectin, including reducingor abrogating the activity of at least one of its ligand-binding sites.Suitably, the E-selectin antagonist reduces the expression of theE-selectin gene or the level or functional activity of an expressionproduct of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% relative to the expression, level or functional activity in theabsence of the agent. In some embodiments, the E-selectin antagonist isa selective E-selectin antagonist.

In accordance with the present invention, E-selectin antagonists areuseful for enhancing a hematopoietic function (e.g., increasing thenumber of hematopoietic stem cells or progenitor cells and/orneutrophils in the peripheral blood) of a mobilizer of hematopoieticstem cells or progenitor cells. The E-selectin antagonists may be knownor identified using any suitable screening assay. Accordingly, in arelated aspect, the present invention provides screening methods foridentifying agents that are useful for enhancing a hematopoieticfunction of the mobilizer. In some embodiments, the screening methodscomprise contacting a preparation with a test agent, wherein thepreparation comprises (i) a polypeptide comprising an amino acidsequence corresponding to at least a biologically active fragment of anE-selectin polypeptide, or to a variant or derivative thereof; or (ii) apolynucleotide comprising at least a portion of a genetic sequence(e.g., a transcriptional element) that regulates the expression of anE-selectin gene, which is operably linked to a reporter gene. A detectedreduction in the level and/or functional activity of the polypeptide, oran expression product of the reporter gene, relative to a normal orreference level and/or functional activity in the absence of the testagent, indicates that the agent is useful for useful for treating orpreventing the immunocompromised condition. In some embodiments, theE-selectin antagonist antagonizes the binding between E-selectin and anE-selectin ligand, as determined by: contacting an E-selectin and theligand with the agent and measuring the binding of the E-selectin withthe ligand. In these embodiments, agents can bind to the E-selectin orto the ligand and test positive when they reduce or abrogate the bindingof the E-selectin with the ligand.

In another related aspect, the present invention provides methods ofproducing an agent that enhances a hematopoietic function of a mobilizerof hematopoietic stem cells or progenitor cells. These methods generallycomprise: testing an agent suspected of antagonizing the function ofE-selectin as broadly described above; and synthesizing the agent on thebasis that it tests positive for the antagonism. Suitably, the methodfurther comprises derivatizing the agent, and optionally formulating thederivatized agent with a pharmaceutically acceptable carrier and/ordiluent, to improve the efficacy of the agent for enhancing thehematopoietic function of the mobilizer.

Another aspect of the present invention provides methods for enhancing ahematopoietic function of a mobilizer of hematopoietic stem cells orprogenitor cells in a subject. These methods generally compriseadministering concurrently to the subject a mobilizer of hematopoieticstem cells or progenitor cells and an E-selectin antagonist in aneffective amount to enhance an hematopoietic function of the mobilizer(e.g., increasing the number of hematopoietic stem cells or progenitorcells and/or neutrophils in the peripheral blood). In a related aspect,the methods are useful for treating or preventing an immunocompromisedcondition in a subject, which condition results from exposure of thesubject to a medical treatment. In these embodiments, the mobilizer andthe E-selectin antagonist are administered in amounts effective fortreatment or prevention the immunocompromised condition. Suitably, theimmunocompromised condition is selected from neutropenia,agranulocytosis, thrombocytopenia, and anemia. In some embodiments, themethods further comprise identifying a subject having or at risk ofdeveloping the immunocompromised condition.

In some embodiments, the medical treatment targets rapidly dividingcells or disrupts the cell cycle or cell division. In illustrativeexamples of this type, the medical treatment is selected fromchemotherapy and radiation therapy. Suitably, the medical treatmentcomprises treatment or prophylaxis of a cancer (e.g., a primary canceror a metastatic cancer) or an autoimmune disease.

The mobilizer and the E-selectin antagonist are suitably administered inthe form of one or more compositions each comprising a pharmaceuticallyacceptable carrier and/or diluent. The composition(s) may beadministered by injection, by topical application or by the oral routeincluding sustained-release modes of administration, over a period oftime and in amounts which are effective for increasing the number ofhematopoietic stem cells or progenitor cells and/or neutrophils in theperipheral blood.

In some embodiments, the mobilizer and the antagonist are administeredsimultaneously to the subject. In other embodiments the E-selectinantagonist is administered to the subject prior to administration of themobilizer. In still other embodiments, the E-selectin antagonist isadministered after administration of the mobilizer to the subject.

Similarly, the E-selectin antagonist and the mobilizer may beadministered to the subject simultaneously, sequentially or separatelywith the medical treatment. In some embodiments, the concurrentadministration of the E-selectin antagonist and the mobilizer is aprophylactic treatment (e.g., the subject is preparing to undergochemotherapy or radiation treatment). In others, it is a therapeutictreatment (e.g., the subject has received at least one dose ofchemotherapy or at least one radiation treatment).

In some embodiments, the methods may further comprise exposing thesubject to an ancillary treatment that treats or prevents theimmunocompromised condition. In illustrative examples of this type, theimmunocompromised condition is anemia and the ancillary treatment maycomprise administering to the subject an anemia medicament selected fromrecombinant erythropoietin (EPO), ferrous iron, ferric iron, vitaminB12, vitamin B6, vitamin C, vitamin D, calcitriol, alphacalcidol,folate, androgen, and carnitine. In other illustrative examples, theimmunocompromised condition is thrombocytopenia and the ancillarytreatment may comprise administering to the subject a thrombocytopeniamedicament selected from a glucocorticoid, recombinant thrombopoietin(TPO), recombinant megakaryocyte growth and development factor (MGDF),pegylated recombinant MGDF and lisophylline. In still other illustrativeexamples, the immunocompromised condition is neutropenia and theancillary treatment suitably comprises administering to the subject aneutropenia medicament selected from glucocorticoid, immunoglobulin,androgens, recombinant IFN-γ, and uteroferrin. In some embodiments, theancillary treatment is administered to the subject simultaneously,sequentially or separately with the E-selectin antagonist and/or themobilizer.

In some embodiments, the medical treatment is likely to expose thesubject to a higher risk of infection. Accordingly, in theseembodiments, the methods may further comprise administeringsimultaneously, sequentially or separately with the E-selectinantagonist and/or the mobilizer at least one anti-infective agent thatis effective against an infection that develops or that has an increasedrisk of developing from the immunocompromised condition, wherein theanti-infective is selected from antimicrobials, antibiotics, antivirals,antifungals, anthelmintics, antiprotozoals and nematocides.

Typically, one or both of the E-selectin antagonist and the mobilizerare administered on a routine schedule, for example, every day, at leasttwice a week, at least three times a week, at least four times a week,at least five times a week, at least six times a week, every week, everyother week, every third week, every fourth week, every month, every twomonths, every three months, every four months, and every six months.

In some advantageous embodiments, the concurrent administration of anE-selectin antagonist and a mobilizer of hematopoietic stem cells orprogenitor cells (also referred to herein as the combination therapy ofthe present invention) is useful for treating or preventinghematopoeitic disorders such as neutropenia, agranulocytosis,thrombocytopenia, and anemia, which may result, for example, frommyelosuppressive treatments that target rapidly dividing cells or thatdisrupt the cell cycle or cell division (e.g., chemotherapy or radiationtherapy). It is proposed, therefore, that since administration of thecombination therapy will reduce the risk of having or developing ahematopoietic disorder as a side effect of the myelosuppressivetreatment, it is possible to administer higher therapeutic doses of achemotherapeutic agent or radiation to a subject in order to kill orinhibit the growth or proliferation of a tumor or to treat or prevent anautoimmune disease in the subject. Accordingly, in yet another aspect,the present invention provides methods for increasing the dose of amedicament in a subject, wherein the medicament results or increases therisk of developing an immunocompromised condition. These methodsgenerally comprise administering concurrently the medicament to thesubject in a dose that ordinarily induces side effects (e.g., thedevelopment of the immunocompromised condition), together with amobilizer of hematopoietic stem cells or progenitor cells and anE-selectin antagonist in amounts effective for inhibiting or preventingthe induction of those side effects (e.g., in amounts effective forincreasing the number of hematopoietic stem cells or progenitor cellsand/or neutrophils in the peripheral blood).

In yet another aspect, the present invention provides pharmaceuticalcompositions for treating or preventing a disease (e.g., cancer or anautoimmune disease) that is treatable or preventable by a medicaltreatment that targets rapidly dividing cells or that disrupts the cellcycle or cell division (e.g., chemotherapy or radiation therapy). Thesecompositions generally comprise an E-selectin antagonist, a mobilizer ofhematopoietic stem cells or progenitor cells and at least one otheragent selected from a chemotherapeutic agent (e.g., a cytotoxic agent),a radiosensitizing agent, an anemia medicament, a thrombocytopeniamedicament, a neutropenia medicament, an agranulocytosis medicament andan anti-infective agent.

Still another aspect of the present invention provides the use of anE-selectin antagonist for enhancing a hematopoietic function of amobilizer of hematopoietic stem cells or progenitor cells.

In yet another aspect, the present invention provides the use of anE-selectin antagonist and a mobilizer of hematopoietic stem cells orprogenitor cells for treating or preventing an immunocompromisedcondition that results from a medical treatment. In some embodiments,the E-selectin antagonist and optionally the mobilizer are prepared ormanufactured as medicaments for this purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical and photographic representation showing thekinetics of BrdU incorporation in vivo in hematopoietic stem cellsisolated form the bone marrow of mice lacking P-selectin and/orE-selectin genes. The top panel shows the percentage of bone marrowLSK34− hematopoietic stem cells positive for BrdU in different knock-outstrains and wild-type mice. The bottom panel shows a typical micrographof BrdU staining (brown colour) in hematopoietic stem cells isolatedfrom bone marrow of wild-type and P/E-selectin double knock-out mice fedfor 5 days with BrdU.

FIG. 2 is a graphical representation showing that LSK hematopoietic stemcells are less metabolically active in E-selectin KO mice than wild-typemice. Upper panels show the gating strategy to measure rhodamine 123incorporation into LSK hematopoietic stem cells. The bottom panel showsthe percentage of bone marrow LSK hematopoietic stem cells thatincorporate low levels of Rhodamine123.

FIG. 3 is a graphical representation showing lower HSC turn-over in bonemarrow of E-selectin KO mice following chemotherapy with the cytotoxicdrug 5FU. These data represent the percentage of cyclinglineage-negative Sca1-positive CD41-negative CD48-negativeCD150-positive long-term reconstituting hematopoietic stem cells thatincorporated BrdU over a period of 17 hours of continuous administrationof BrdU prior sacrifice at days 3 (left panel) and day 7 (right panel)following administration of a single dose of 150 mg/kg of 5FU. Each dotrepresents result from an individual mouse. The horizontal bar is theaverage of the group.

FIG. 4 is a graphical representation showing higher number of long-termreconstituting hematopoietic stem cells in E-selectin KO bone marrowfollowing chemotherapy with the cytotoxic drug 5-FU. These datarepresent the total number of lineage-negative Sca1-positiveCD41-negative CD48-negative CD 150-positive long-term reconstitutinghematopoietic stem cells per femur at the indicated time-pointsfollowing administration of a single dose of 150 mg/kg of 5FU. Each dotrepresents result from an individual mouse. The horizontal bar is theaverage of the group.

FIG. 5 is a graphical representation showing that deletion of the PSGL1and CD44 genes has no effect on adhesion of hematopoietic progenitorcells to recombinant mouse E-selectin immobilised to plastic.Recombinant muE-selectin-IgG1Fc, muP-selectin-IgG1Fc and muVCAM1-IgG1Fcwere adsorbed to the bottom of 96-well polystyrene tissue culture platesovernight, then the wells thoroughly washed and blocked before theaddition of 30,000 calcein-labelled Lineage-negative CD117-positivecells from sorted from the bone marrow of wild-type, PSGL-1−/−, CD44−/−,or CD44−/−/PSGL−/− double KO mice. Data are expressed as a percentage ofadherent cells and a means+/− standard deviation of triplicates.

FIG. 6 is a graphical representation showing that deletion of CD44 andPSGL1 genes does not perturb binding of soluble recombinantE-selectin-IgMFc fusion protein to the surface of LSK hematopoietic stemcells. Top panels show the gating strategy to measure selectin-IgMbinding at the surface of bone marrow CD11b-positive Gr1-positivegranulocytes and lineage-negative Sca1-positive CD117-positive (LSK)hematopoietic stem cells. The bottom panels show the proportion of thesebinding either the recombinant E-selectin-IgMFc or P-selectin-IgMFcfusion proteins.

FIG. 7 is a graphical representation showing increased mobilization ofCFC, phenotypic HSC and functional HSC in E-selectin KO mice in responseto G-CSF. Groups of mice, either wild-type C57BL/6 or E-selectinknockout on a C57BL/6 background were mobilized according to theprotocol described in the Materials and Methods section below(administration of a total 1 mg/kg pegylated recombinant human G-CSF)and blood collected 3 days later for analysis of mobilized HSC content.A second cohort of mice received saline injections and are designated asnon-mobilized controls. Blood was analyzed for total leukocyte count(7A), colony forming cells (7B), phenotypic HSC (7C) and a portioninjected into lethally irradiated recipient mice to determine the numberof repopulating units (RU) in a long-term competitive reconstitutionassay (7D). Significant levels are indicated (p<0.05) and werecalculated using the non-parametric Mann-Whitney test.

FIG. 8 is a graphical representation showing increased mobilization ofCFC and phenotypic HSC in E-selectin KO mice which underwent 8 cycles ofchemotherapy with cyclophosphamide prior to mobilization with G-CSF.Wild-type and E-selectin KO mice were first administered fortnightlyrounds of the chemotherapeutic agent cyclophosphamide at 200 mg/kg for 8rounds, then allowed to rest for 6 weeks prior to administration ofG-CSF (total 1 mg/kg as above) and blood collected 3 days when HSC aremobilised. Blood was analyzed for total leukocyte count (8A), Colonyforming cells (8B) and phenotypic HSC (8C).

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “administration concurrently” or “administering concurrently”or “co-administering” and the like refer to the administration of asingle composition containing two or more actives, or the administrationof each active as separate compositions and/or delivered by separateroutes either contemporaneously or simultaneously or sequentially withina short enough period of time that the effective result is equivalent tothat obtained when all such actives are administered as a singlecomposition. For example, an E-selectin antagonist may be administeredtogether with a mobilizer of hematopoietic stem cells or progenitorcells in order to increase the numbers of hematopoietic stem cells,progenitor cells and/or granulocytes (e.g., neutrophils) in peripheralblood. In another example, an E-selectin antagonist and a mobilizer ofhematopoietic stem cells or progenitor cells are administered togetherwith another agent to enhance their effects or to ameliorate the effectsof a medical treatment that causes or contributes to animmunocompromised condition. By “sequential” administration is meant atime difference of from seconds, minutes, hours or days between theadministration of the two types of molecules or active agents. Thesemolecules or active agents may be administered in any order. By“simultaneously” is meant that the active agents are administered atsubstantially the same time, and desirably together in the sameformulation. By “contemporaneously” it is meant that the active agentsare administered closely in time, e.g., one agent is administered withinfrom about one minute to within about one day before or after another.Any contemporaneous time is useful. However, it will often be the casethat when not administered simultaneously, the agents will beadministered within about one minute to within about eight hours andpreferably within less than about one to about four hours. In certainembodiments, the E-selectin antagonist and the mobilizer areadministered within about 60 minutes, about 50 minutes, about 40minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5minutes, or about 1 minute of each other or separated in time by about 1hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about12 hours, about 24 hours, about 36 hours, or about 72 hours, or more.When administered contemporaneously, the agents are suitablyadministered at the same site on the subject. The term “same site”includes the exact location, but can be within about 0.5 to about 15centimeters, usually from within about 0.5 to about 5 centimeters. Theterm “separately” as used herein means that the agents are administeredat an interval, for example at an interval of about a day to severalweeks or months. The active agents may be administered in either order.The term “sequentially” as used herein means that the agents areadministered in sequence, for example at an interval or intervals ofminutes, hours, days or weeks. If appropriate the active agents may beadministered in a regular repeating cycle.

The term “agent” or “modulatory agent” includes a compound that inducesa desired pharmacological and/or physiological effect. The term alsoencompass pharmaceutically acceptable and pharmacologically activeingredients of those compounds specifically mentioned herein includingbut not limited to salts, esters, amides, prodrugs, active metabolites,analogs and the like. When the above term is used, then it is to beunderstood that this includes the active agent per se as well aspharmaceutically acceptable, pharmacologically active salts, esters,amides, prodrugs, metabolites, analogs, etc. The term “agent” is not tobe construed narrowly but extends to small molecules, proteinaceousmolecules such as peptides, polypeptides and proteins as well ascompositions comprising them and genetic molecules such as RNA, DNA andmimetics and chemical analogs thereof as well as cellular agents. Theterm “agent” includes a cell which is capable of producing and secretingthe polypeptides referred to herein as well as a polynucleotidecomprising a nucleotide sequence that encodes this polypeptide. Thus,the term “agent” extends to nucleic acid constructs including vectorssuch as viral or non-viral vectors, expression vectors and plasmids forexpression in and secretion in a range of cells.

An “agranulocytosis medicament” as used herein refers to a compositionof matter which reduces the symptoms related to agranulocytosis,prevents the development of agranulocytosis, or treats existingagranulocytosis.

An “anemia medicament” as used herein refers to a composition of matterwhich reduces the symptoms related to anemia, prevents the developmentof anemia, or treats existing anemia.

As used herein, the term “antagonist” means an agent that decreases orinhibits at least one function or biological activity of E-selectin(also known as CD62E, ELAM-1, LECAM-2). An E-selectin antagonist may bea compound which inhibits or decreases the interaction betweenE-selectin and another molecule, e.g., a target peptide, polypeptide,receptor, ligand or enzyme substrate. An antagonist may also be acompound that down-regulates expression of an E-selectin gene or whichreduces the amount of expressed E-selectin protein present.

By “antigen-binding molecule” is meant a molecule that has bindingaffinity for a target antigen. It will be understood that this termextends to immunoglobulins, immunoglobulin fragments andnon-immunoglobulin derived protein frameworks that exhibitantigen-binding activity.

“Antigenic or immunogenic activity” refers to the ability of apolypeptide, fragment, variant or derivative according to the inventionto produce an antigenic or immunogenic response in an animal, suitably amammal, to which it is administered, wherein the response includes theproduction of elements which specifically bind the polypeptide orfragment thereof.

Reference herein to “bacteria” or “bacterial infection” includes anybacterial pathogen including emerging bacterial pathogen of vertebrates.Representative bacterial pathogens include without limitation speciesof: Acinetobacter, Actinobacillus, Actinomycetes, Actinomvces,Aeromonas, Bacillus, Bacteroides, Bordetella, Borrelia, Brucella(brucellosis), Burkholderia, Campylobacter, Citrobacter, Clostridium,Corvnebacterium, Enterobacter, Enterococcus, Erysipelolhrix,Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella,Legionella, Leptospira, Listeria, Micrococcus, Moraxella, Morganella,Mvcobacterium (tuberculosis), Nocardia, Neisseria, Pasteurella,Plesiomonas, Propionibacterium, Proteus, Providencia, Pseudomonas,Rhodococcus, Salmonella, Serratia, Shigella. Staphylococcus,Stenotrophomonas, Streptococcus, Treponema, Vibrio (cholera) andYersinia (plague).

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “corresponds to” or “corresponding to” is meant (a) a polynucleotidehaving a nucleotide sequence that is substantially identical orcomplementary to all or a portion of a reference polynucleotide sequenceor encoding an amino acid sequence identical to an amino acid sequencein a peptide or protein; or (b) a peptide or polypeptide having an aminoacid sequence that is substantially identical to a sequence of aminoacids in a reference peptide or protein.

By “derivative” is meant a polypeptide that has been derived from thebasic sequence by modification, for example by conjugation or complexingwith other chemical moieties or by post-translational modificationtechniques as would be understood in the art. The term “derivative” alsoincludes within its scope alterations that have been made to a parentsequence including additions or deletions that provide for functionalequivalent molecules.

The term “differentiation” of hematopoietic stem cells and/orhematopoietic progenitors as used herein refers to both the change ofhematopoietic stem cells into hematopoietic progenitors and the changeof hematopoietic progenitors into unipotent hematopoietic progenitorsand/or cells having characteristic functions, namely mature cellsincluding erythrocytes, leukocytes (e.g., neutrophils) andmegakaryocytes. Differentiation of hematopoietic stem cells into avariety of blood cell types involves sequential activation or silencingof several sets of genes. Hematopoietic stem cells typically chooseeither a lymphoid or myeloid lineage pathway at an early stage ofdifferentiation.

By “effective amount,” is meant the administration of an amount ofactive agent to a subject, either in a single dose or as part of aseries or slow release system, which is effective for prevention ortreatment. The effective amount will vary depending upon the health andphysical condition of the subject and the taxonomic group of individualto be treated, the formulation of the composition, the assessment of themedical situation, and other relevant factors.

As used herein, the term “function” refers to a biological, enzymatic,or therapeutic function.

The terms “expression” or “gene expression” refer to either productionof RNA message or translation of RNA message into proteins orpolypeptides. By “expression vector” is meant any genetic elementcapable of directing the transcription of a polynucleotide containedwithin the vector and suitably the synthesis of a peptide or polypeptideencoded by the polynucleotide. Such expression vectors are known topractitioners in the art.

The term “gene” as used herein refers to any and all discrete codingregions of the cell's genome, as well as associated non-coding andregulatory regions. The term is intended to mean the open reading frameencoding specific polypeptides, introns, and adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression. In this regard, the gene may further comprise controlsignals such as promoters, enhancers, termination and/or polyadenylationsignals that are naturally associated with a given gene, or heterologouscontrol signals. The DNA sequences may be cDNA or genomic DNA or afragment thereof. The gene may be introduced into an appropriate vectorfor extrachromosomal maintenance or for integration into the host.

“Hematopoiesis” refers to the highly orchestrated process of blood celldevelopment and homeostasis. Prenatally, hematopoiesis occurs in theyolk sack, then liver, and eventually the bone marrow. In normal adultsit occurs in bone marrow and lymphatic tissues. All blood cells developfrom pluripotent stem cells. Pluripotent cells differentiate into stemcells that are committed to three, two or one hematopoieticdifferentiation pathway. None of these stem cells are morphologicallydistinguishable, however.

The term “hematopoietic stem cells” as used herein refers to multipotentstem cells that are capable of differentiating into all blood cellsincluding erythrocytes, leukocytes and platelets. For instance, the term“hematopoietic stem cells” includes and encompasses those contained notonly in bone marrow but also in umbilical cord blood derived cells.

The term “hematopoietic progenitors,” which is used interchangeably withthe term “hematopoietic precursors,” refers to those progenitor orprecursor cells which are differentiated further than hematopoietic stemcells but have yet to differentiate into progenitors or precursors ofrespective blood cell lineages (unipotent precursor cells). Thus,“progenitor cell(s)” or “precursor cell(s)” are defined as cells thatare lineage-committed, i.e., an individual cell can give rise to progenylimited to a single lineage such as the myeloid or lymphoid lineage.They do not have self-renewal properties. They can also be stimulated bylineage-specific growth factors to proliferate. If activated toproliferate, progenitor cells have life-spans limited to 50-70 celldoublings before programmed cell senescence and death occurs. Forexample, “hematopoietic progenitors” as used herein includegranulocyte/macrophage associated progenitors (colony-forming unitgranulocyte, macrophage, CFU-GM), erythroid associated progenitors(burst-forming unit erythroid, BFU-E), megakaryocyte associatedprogenitors (colony-forming unit megakaryocyte, CFU-Mk), and myeloidassociated stem cells (colony-forming unit mixed, CFU-Mix).Hematopoietic progenitor cells possess the ability to differentiate intoa final cell type directly or indirectly through a particulardevelopmental lineage. Undifferentiated, pluripotent progenitor cellsthat are not committed to any lineage are referred to herein as “stemcells.” All hematopoietic cells can in theory be derived from a singlestem cell, which is also able to perpetuate the stem cell lineage, asdaughter cells become differentiated. The isolation of populations ofmammalian bone marrow cell populations which are enriched to a greateror lesser extent in pluripotent stem cells has been reported (see forexample, C. Verfaillie el al., J. Exp. Med., 172. 509 (1990),incorporated herein by reference).

“Homolog” is used herein to denote a gene or its product which isrelated to another gene or product by decent from a common ancestral DNAsequence.

“Hybridization” is used herein to denote the pairing of complementarynucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.Complementary base sequences are those sequences that are related by thebase-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA Upairs with A and C pairs with G. In this regard, the terms “match” and“mismatch” as used herein refer to the hybridization potential of pairednucleotides in complementary nucleic acid strands. Matched nucleotideshybridize efficiently, such as the classical A-T and G-C base pairmentioned above. Mismatches are other combinations of nucleotides thatdo not hybridize efficiently. In the present invention, the preferredmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances as known tothose of skill in the art.

The phrase “hybridizing specifically to” and the like refer to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “immunocompromised” as used herein refers to a subject with aninnate, acquired, or induced inability to develop a normal immuneresponse. An immunocompromised subject, therefore, has a weakened orimpaired immune system relative to one of a normal subject. A subjectwith a weakened or impaired immune system has an “immunodeficiency” or“immunocompromised condition.” which is associated with a primary orsecondary deficiency, induced or non-induced, in one or more of theelements of the normal immune defense system. An immunocompromisedcondition is commonly due to a medical treatment, e.g., radiationtherapy, chemotherapy or other immunosuppressing treatment, such asinduced by treatment with steroids, cyclophosphamide, azathioprine,methotrexate, cyclosporine or rapamycin, in particular in relation tocancer treatment or the treatment or prevention of transplant rejection.However, it will be understood that the phrase “risk of acquiring animmunocompromised condition resulting from a medical treatment” refersonly to medical treatments that leads to or confers an immunocompromisedcondition, especially chemotherapy or other immunosuppressing treatment,such as induced by treatment with radiation, steroids, cyclophosphamide,azathioprine, methotrexate, cyclosporine or rapamycin. The presence ofan immunocompromised condition in a subject can be diagnosed by anysuitable technique known to persons of skill the art. Strong indicatorsthat an immunocompromised condition may be present is when rare diseasesoccur or the subject gets ill from organisms that do not normally causediseases, especially if the subject gets repeatedly infected. Otherpossibilities are typically considered, such as recently acquiredinfections—for example, HIV, hepatitis, tuberculosis, etc. Generally,however, definitive diagnoses are based on laboratory tests thatdetermine the exact nature of the immunocompromised condition. Mosttests are performed on blood samples. Blood contains antibodies,lymphocytes, phagocytes, and complement components—all of the majorimmune components that might cause immunodeficiency. A blood cell countwill determine if the number of phagocytic cells or lymphocytes is belownormal. Lower than normal counts of either of these two cell typescorrelates with an immunocompromised condition. The blood cells are alsochecked for their appearance. Occasionally, a subject may have normalcell counts, but the cells are structurally defective. If the lymphocytecell count is low, further testing is usually conducted to determinewhether any particular type of lymphocyte is lower than normal. Alymphocyte proliferation test may be conducted to determine if thelymphocytes can respond to stimuli. The failure to respond to stimulantscorrelates with an immunocompromised condition. Antibody levels andcomplement levels can also be determined for diagnosing the presence ofan immunocompromised condition. However, it shall be understood that themethods of the present invention are not predicated upon diagnosing theabsence of an immunocompromised condition in the subjects to be treated.

Reference herein to “immuno-interactive” includes reference to anyinteraction, reaction, or other form of association between moleculesand in particular where one of the molecules is, or mimics, a componentof the immune system.

Reference herein to a “infectious agent,” “infectious organism,”“microbe” or “pathogen” includes any one or more species or subspeciesof bacterium, fungus, virus, algae, parasite, (including ecto- orendo-parasites) prion, oomycetes, slime, moulds, nematodes, mycoplasmaand the like. The present invention is particularly suited to treatingor preventing mixed infections by more than one microbe. Pathogenicalgae include Prototheca and Pfiesteria. Also includes within the scopeof these terms are prion proteins causing conditions such asCreutzfeldt-Jakob disease. As the skilled artisan will appreciate,pathogenicity or the ability of a classically non-pathogenic agent toinfect a subject and cause pathology can vary with the genotype andexpression profile of the infectious agent, the host and theenvironment. Fungal pathogens include without limitation species of thefollowing genera: Absidia, Acremonium, Aspergillus. Basidiobolus,Bipolaris, Blastomyces, Candida (yeast), Cladophialophora, Coccidioides,Criptococcus, Cunninghamella, Curvularia, Epidermophyton, Exophiala,Exserohilum, Fonsecaea, Fusarium, Geotrichum, Histoplasma, Hortaea,Lacazia, Lasiodiplodia, Leptosphaeria, Madurella, Malassezia,Microsporum, Mucor, Neotestudina, Onvchocola, Paecilomyces,Paracoccidioides, Penicillium, Phialophora, Piedraia, Piedra,Pityriasis, Pneumocystis, Pseudallescheria, Pyrenochaeta, Rhizomucor,Rhizopus, Rhodotorula, Scedosporium, Scopulariopsis, Scytalidium,Sporothrix, Trichophyton, Trichosporon and Zygomycete. Pathogenicconditions include any deleterious condition that develops as a resultof infection with an infectious organism.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state.

The term “lower alkyl” refers to straight and branched chain alkylgroups having from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl,iso-propyl, n-butyl, ten-butyl, sec-butyl, n-pentyl, n-hexyl,2-methylpentyl, and the like. In some embodiments, the lower alkyl groupis methyl or ethyl.

The term “lower alkoxy” refers to straight and branched chain alkoxygroups having from 1 to 6 carbon atoms, such as methoxy, ethoxy,n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy,n-hexoxy, 2-methyl-pentoxy, and the like. Usually, the lower alkoxygroup is methoxy or ethoxy.

As used herein, a “mobilizer of hematopoietic stem cells or progenitorcells,” “mobilizing agent” or “mobilizer” are used interchangeably torefer to any compound, whether it is a small organic molecule, syntheticor naturally derived, or a polypeptide, such as a growth factor orcolony stimulating factor or an active fragment or mimic thereof, anucleic acid, a carbohydrate, an antibody, or any other agent that actsto enhance the migration of stem cells from the bone marrow into theperipheral blood. Such a “mobilizer” may increase the number ofhematopoietic stem cells or hematopoietic progenitor/precursor cells inthe peripheral blood.

By “modulating” is meant increasing or decreasing, either directly orindirectly, the level or functional activity of a target molecule. Forexample, an agent may indirectly modulate the level/activity byinteracting with a molecule other than the target molecule. In thisregard, indirect modulation of a gene encoding a target polypeptideincludes within its scope modulation of the expression of a firstnucleic acid molecule, wherein an expression product of the firstnucleic acid molecule modulates the expression of a nucleic acidmolecule encoding the target polypeptide.

A “neutropenia medicament” as used herein refers to a composition ofmatter which reduces the symptoms related to neutropenia, prevents thedevelopment of neutropenia, or treats existing neutropenia.

The term “oligonucleotide” as used herein refers to a polymer composedof a multiplicity of nucleotide residues (deoxyribonucleotides orribonucleotides, or related structural variants or synthetic analoguesthereof) linked via phosphodiester bonds (or related structural variantsor synthetic analogues thereof). Thus, while the term “oligonucleotide”typically refers to a nucleotide polymer in which the nucleotideresidues and linkages between them are naturally occurring, it will beunderstood that the term also includes within its scope variousanalogues including, but not restricted to, peptide nucleic acids(PNAs), phosphoramidates, phosphorothioates, methyl phosphonates,2-O-methyl ribonucleic acids, and the like. The exact size of themolecule can vary depending on the particular application. Anoligonucleotide is typically rather short in length, generally fromabout 10 to 30 nucleotide residues, but the term can refer to moleculesof any length, although the term “polynucleotide” or “nucleic acid” istypically used for large oligonucleotides.

By “operably linked” is meant that transcriptional and translationalregulatory polynucleotides are positioned relative to apolypeptide-encoding polynucleotide in such a manner that thepolynucleotide is transcribed and the polypeptide is translated.

By “pharmaceutically acceptable carrier” is meant a pharmaceuticalvehicle comprised of a material that is not biologically or otherwiseundesirable, i.e. the material may be administered to a subject alongwith the selected active agent without causing any or a substantialadverse reaction. Carriers may include excipients and other additivessuch as diluents, detergents, coloring agents, wetting or emulsifyingagents, pH buffering agents, preservatives, and the like.

Similarly, a “pharmacologically acceptable” salt, ester, amide, prodrugor derivative of a compound as provided herein is a salt, ester, amide,prodrug or derivative that this not biologically or otherwiseundesirable.

Pathogenic “protozoa” include, without limitation, Trypanosoma,Leishmania, Giardia, Trichomonas, Entamoeba, Naegleria, Acanthamoeba,Plasmodium, Toxoplasma, Cryptosporidium, Isospora and Balanlidium.

Larger pathogenic “parasites” include those from the phyla Cestoda(Lapeworms), Nematoda and Trematoda (flukes). Pathogenic trematodes are,for example, species of the following genera; Schistosoma, Echinostoma,Fasciolopsis, Clonorchis, Fasciola, Opisthorchis and Paragonimus.Cestode pathogens include, without limitation, species from thefollowing orders; Pseudophyllidea (e.g., Diphyllobothrium) andCyclophyllidea (e.g., Taenia). Pathogenic nematodes include species fromthe orders; Rhabditida (e.g., Strongyloides), Strongylida (e.g.,Ancylostoma), Ascaridia (e.g., Ascaris, Toxocara), Spirurida (e.g.,Dracunculus, Brugia, Onchocerca, Wucheria) and Adenophorea(e.g.,Trichuris and Trichinella).

The terms “polynucleotide,” “genetic material,” “genetic forms.”“nucleic acids” and “nucleotide sequence” include RNA, cDNA, genomicDNA, synthetic forms and mixed polymers, both sense and antisensestrands, and may be chemically or biochemically modified or may containnon-natural or derivatized nucleotide bases, as will be readilyappreciated by those skilled in the art.

The terms “polynucleotide variant” and “variant” refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridise witha reference sequence under stringent conditions as known in the art (seefor example Sambrook el al., Molecular Cloning. A Laboratory Manual”,Cold Spring Harbor Press, 1989). These terms also encompasspolynucleotides in which one or more nucleotides have been added ordeleted, or replaced with different nucleotides. In this regard, it iswell understood in the art that certain alterations inclusive ofmutations, additions, deletions and substitutions can be made to areference polynucleotide whereby the altered polynucleotide retains abiological function or activity of the reference polynucleotide. Theterms “polynucleotide variant” and “variant” also includenaturally-occurring allelic variants.

The terms “polypeptide,” “proteinaceous molecule,” “peptide” and“protein” are used interchangeably herein to refer to a polymer of aminoacid residues and to variants and synthetic analogues of the same. Thus,these terms apply to amino acid polymers in which one or more amino acidresidues is a synthetic non-naturally-occurring amino acid, such as achemical analogue of a corresponding naturally-occurring amino acid, aswell as to naturally-occurring amino acid polymers. These terms do notexclude modifications, for example, glycosylations, acetylations,phosphorylations and the like. Soluble forms of the subjectproteinaceous molecules are particularly useful. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid including, for example, unnatural amino acids orpolypeptides with substituted linkages.

The term “polypeptide variant” refers to polypeptides in which one ormore amino acids have been replaced by different amino acids. It is wellunderstood in the art that some amino acids may be changed to otherswith broadly similar properties without changing the nature of theactivity of the polypeptide (conservative substitutions) as describedhereinafter. These terms also encompass polypeptides in which one ormore amino acids have been added or deleted, or replaced with differentamino acids.

As used herein, the terms “prevent,” “prevented,” or “preventing,” whenused with respect to the treatment of an immunocompromised condition(e.g., anemia, thrombocytopenia, agranulocytosis or neutropenia), refersto a prophylactic treatment which increases the resistance of a subjectto developing the immunocompromised condition or, in other words,decreases the likelihood that the subject will develop theimmunocompromised condition as well as a treatment after theimmunocompromised condition has begun in order to reduce or eliminate italtogether or prevent it from becoming worse.

As used herein; a “reporter gene” refers to any gene or DNA thatexpresses a product that is detectable by spectroscopic, photochemical,biochemical, enzymatic, immunochemical, electrical, optical or chemicalmeans. The preferred reporter gene to which a promoter element isligated is luciferase. Other reporter genes for use for this purposeinclude, for example, β-galactosidase gene (β-gal) and chloramphenicolacetyltransferase gene (CAT) Assays for expression produced inconjunction with each of these reporter gene elements are well-known tothose skilled in the art.

The term “selective” refers to compounds that inhibit or displayantagonism towards E-selectin without displaying substantial inhibitionor antagonism towards another selectin (e.g., P-selectin). Accordingly,a compound that is selective for E-selectin exhibits an E-selectinselectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold,50-fold or greater than about 100-fold with respect to inhibition orantagonism of another selectin (i.e., a selectin other than E-selectin).In some embodiments, selective compounds display at least 50-foldgreater inhibition or antagonism towards E-selectin than towardsP-selectin. In still other embodiments, selective compounds inhibit ordisplay at least 100-fold greater inhibition or antagonism towardsE-selectin than towards P-selectin. In still other embodiments,selective compounds display at least 500-fold greater inhibition orantagonism towards E-selectin than towards P-selectin. In still otherembodiments, selective compounds display at least 1000-fold greaterinhibition or antagonism towards E-selectin than towards P-selectin.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by anappropriate method. For example, sequence identity analysis may becarried out using the DNASIS computer program (Version 2.5 for windows;available from Hitachi Software engineering Co., Ltd., South SanFrancisco, Calif., USA) using standard defaults as used in the referencemanual accompanying the software.

“Similarity” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions as defined in Table Abelow. Similarity may be determined using sequence comparison programssuch as GAP (Deveraux et al. 1984, Nucleic Acids Research 12, 387-395).In this way, sequences of a similar or substantially different length tothose cited herein might be compared by insertion of gaps into thealignment, such gaps being determined, for example, by the comparisonalgorithm used by GAP. Terms used to describe sequence relationshipsbetween two or more polynucleotides or polypeptides include “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity” and “substantial identity”. A “reference sequence” isat least 12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology”, John Wiley & Sons Inc, 1994-1998, Chapter 15.

As used herein a “small molecule” refers to a composition that has amolecular weight of less than 3 kilodaltons (kDa), and typically lessthan 1.5 kilodaltons, and more preferably less than about 1 kilodalton.Small molecules may be nucleic acids, peptides, polypeptides,peptidomimetics, carbohydrates, lipids or other organic(carbon-containing) or inorganic molecules. As those skilled in the artwill appreciate, based on the present description, extensive librariesof chemical and/or biological mixtures, often fungal, bacterial, oralgal extracts, may be screened with any of the assays of the inventionto identify compounds that modulate a bioactivity. A “small organicmolecule” is an organic compound (or organic compound complexed with aninorganic compound (e.g., metal)) that has a molecular weight of lessthan 3 kilodaltons, less than 1.5 kilodaltons, or even less than about 1kDa.

“Stem cells” refer to cells, which are not terminally differentiated andare therefore able to produce cells of other types. Stem cells aregenerally divided into three types, including totipotent, pluripotent,and multipotent. “Totipotent stem cells” can grow and differentiate intoany cell in the body, and thus can grow into an entire organism. Thesecells are not capable of self-renewal. In mammals, only the zygote andearly embryonic cells are totipotent. “Pluripotent stem cells” are truestem cells, with the potential to make any differentiated cell in thebody, but cannot contribute to making the extraembryonic membranes(which are derived from the trophoblast). “Multipotent stem cells” areclonal cells that self-renew as well as differentiate to regenerateadult tissues. “Multipotent stem cells” are also referred to as“unipotent” and can only become particular types of cells, such as bloodcells or bone cells. The term “stem cells”, as used herein, refers topluripotent stem cells capable of self-renewal.

“Stringency” as used herein refers to the temperature and ionic strengthconditions, and presence or absence of certain organic solvents, duringhybridization. The higher the stringency, the higher will be theobserved degree of complementarity between sequences. “Stringentconditions” as used herein refers to temperature and ionic conditionsunder which only polynucleotides having a high proportion ofcomplementary bases, preferably having exact complementarity, willhybridize. The stringency required is nucleotide sequence dependent anddepends upon the various components present during hybridization, and isgreatly changed when nucleotide analogues are used. Generally, stringentconditions are selected to be about 10° C. to 20° C. less than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a target sequence hybridizes to a complementaryprobe. It will be understood that a polynucleotide will hybridize to atarget sequence under at least low stringency conditions, preferablyunder at least medium stringency conditions and more preferably underhigh stringency conditions. Reference herein to low stringencyconditions include and encompass from at least about 1% v/v to at leastabout 15% v/v formamide and from at least about 1 M to at least about 2M salt for hybridization at 42° C., and at least about 1 M to at leastabout 2 M salt for washing at 42° C. Low stringency conditions also mayinclude 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2),7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii)0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at roomtemperature. Medium stringency conditions include and encompass from atleast about 16% v/v to at least about 30% v/v formamide and from atleast about 0.5 M to at least about 0.9 M salt for hybridization at 42°C., and at least about 0.5 M to at least about 0.9 M salt for washing at42° C. Medium stringency conditions also may include 1% Bovine SerumAlbumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS forhybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mMEDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 42° C. Highstringency conditions include and encompass from at least about 31% v/vto at least about 50% v/v formamide and from at least about 0.01 M to atleast about 0.15 M salt for hybridization at 42° C., and at least about0.01 M to at least about 0.15 M salt for washing at 42° C. Highstringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4(pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS;or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washingat a temperature in excess of 65° C. Other stringent conditions are wellknown in the art. A skilled addressee will recognize that variousfactors can be manipulated to optimize the specificity of thehybridization. Optimization of the stringency of the final washes canserve to ensure a high degree of hybridization. For detailed examples,see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (supra) at pages 2.10.1 to2.10.16 and MOLECULAR CLONING. A LABORATORY MANUAL (Sambrook, et al.,eds.) (Cold Spring Harbor Press 1989) at sections 1.101 to 1.104.

“Subjects” contemplated in the present invention include any animal ofcommercial humanitarian or epidemiological interest includingconveniently, primates, livestock animals (such as sheep, cows, horses,donkeys, pigs, fish and birds), laboratory test animals (such as mice,rabbits, guinea pigs and hamsters and the like), companion animals (suchas dogs and cats), or captive wild animals. Avian species includepoultry birds and caged avian species. In some embodiments the subjectis a mammalian animal. In other embodiments, the subject is a humansubject. The present composition and methods have applications in humanand veterinary medicine, domestic or wild animal husbandry, cosmetic oraesthetic treatments for the skin after injury or surgery.

By “substantially complementary” it is meant that an oligonucleotide ora subsequence thereof is sufficiently complementary to hybridize with atarget sequence. Accordingly, the nucleotide sequence of theoligonucleotide or subsequence need not reflect the exact complementarysequence of the target sequence. In a preferred embodiment, theoligonucleotide contains no mismatches and with the target sequence.

A “thrombocytopenia medicament” as used herein refers to a compositionof matter which reduces the symptoms related to thrombocytopenia,prevents the development of thrombocytopenia, or treats existingthrombocytopenia.

By “treatment,” “treat,” “treated,” “treating” and the like is meant toinclude both therapeutic and prophylactic treatment, including theadministration of medicine or the performance of medical procedures withrespect to a patient, for either prophylaxis (prevention) or to cure orreduce the extent of or likelihood of occurrence of the infirmity ormalady or condition or event in the instance where the patient isafflicted. In some embodiments of the present invention, the treatmentsusing the agents described may be provided to treat patients sufferingfrom a cancerous condition or hyperproliferative disease, whereby thetreatment of the disease with chemotherapy or irradiation therapyresults in a decrease in bone marrow cellularity, thus making thepatient more immunocompromised and more prone therefore to acquiringinfectious agents or diseases. Thus, the administration of the agents ofthe invention allows for enhanced mobilization of hematopoietic stemcells or progenitor cells from the bone marrow to the peripheral blood.In some embodiments, the treating is for the purpose of reducing ordiminishing the symptoms or progression of a cancerous disease ordisorder by allowing for the use of accelerated doses of chemotherapy orirradiation therapy.

By “vector” is meant a polynucleotide molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, yeast orvirus, into which a polynucleotide can be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and can becapable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector can be an autonomouslyreplicating vector, i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector can contain any means for assuring self-replication.Alternatively, the vector can be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. A vector system cancomprise a single vector or plasmid, two or more vectors or plasmids,which together contain the total DNA to be introduced into the genome ofthe host cell, or a transposon. The choice of the vector will typicallydepend on the compatibility of the vector with the host cell into whichthe vector is to be introduced. In the present case, the vector ispreferably a viral or viral-derived vector, which is operably functionalin animal and preferably mammalian cells. Such vector may be derivedfrom a poxvirus, an adenovirus or yeast. The vector can also include aselection marker such as an antibiotic resistance gene that can be usedfor selection of suitable transformants. Examples of such resistancegenes are known to those of skill in the art and include the nptII genethat confers resistance to the antibiotics kanamycin and G418(Geneticin®) and the hph gene which confers resistance to the antibiotichygromycin B.

Reference herein to “a virus” includes any virus or viral pathogen oremerging viral pathogen. Viral families contemplated includeAdenoviridae, African swine fever-like viruses, Arenaviridae (such asviral haemorrhagic fevers, Lassa fever), Astroviridae (astroviruses)Bunyaviridae (La Crosse), Caliciviridae (Norovirus), Coronaviridae(Corona virus), Filoviridae (such as Ebola virus. Marburg virus),Parvoviridae (B19 virus). Flaviviridae (such as hepatitis C virus,Dengue viruses), Hepadnaviridae (such as hepatitis B virus, Deltavirus),Herpesviridae (herpes simplex virus, varicella zoster virus),Orthomyxoviridae (influenza virus) Papovaviridae (papilloma virus)Paramyxoviridae (such as human parainfluenza viruses, mumps virus,measles virus, human respiratory syncytial virus, Nipah virus, Hendravirus), Picornaviridae (common cold virus), Poxviridae (small pox virus,orf virus, monkey poxvirus) Reoviridae (rotavirus) Retroviridae (humanimmunodeficiency virus) Parvoviridae (parvoviruses) Papillomaviridae,(papillomaviruses) alphaviruses and Rhabdoviridae (rabies virus).

As used herein, underscoring or italicizing the name of a gene shallindicate the gene, in contrast to its protein product, which isindicated by the name of the gene in the absence of any underscoring oritalicizing. For example, “E-selectin” shall mean the E-selecting gene,whereas “E-selectin” shall indicate the protein product or productsgenerated from transcription and translation and alternative splicing ofthe “E-selectin” gene.

2. Abbreviations

CFC=colony-forming cells

HSC=hematopoietic stem cells

d=day

h=hour

s=seconds

i.v.=intravenous

i.p.=intraperitoneal

s.c.=subcutaneous

3. Compositions and Methods for Enhancing Hematopoietic Function

The present invention is based in part on the surprising discovery thatmobilization of hematopoietic stem cells by mobilizing agents such asG-CSF is significantly enhanced in the absence of E-selectin. Thisincreased mobilization in turn results in higher numbers ofhematopoietic stem cells, progenitor cells and granulocytes such asneutrophils in peripheral blood when compared to administration of stemcell mobilizers alone. Accordingly, the present invention relates tomethods and compositions involving concurrent administration of anE-selectin antagonist and a mobilizer of hematopoietic stem cells orprogenitor cells for stimulating or enhancing hematopoiesis and for thetreatment or prophylaxis of immunocompromised conditions resulting frommedical treatments that target rapidly dividing cells or that disruptthe cell cycle or cell division (e.g., myeloablative therapy). Thus, insome embodiments, the present invention provides compositions comprisingan E-selectin antagonist and a mobilizer of hematopoietic stem cells orprogenitor cells.

3.1 E-Selectin Antagonists

The E-selectin antagonist includes and encompasses any active compoundthat binds to E-selectin and that suitably inhibits the functionalactivity of E-selectin, including small molecules, such as nucleicacids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids orother organic (carbon containing) or inorganic molecules. In someembodiments, the E-selectin antagonist is selected from antigen-bindingmolecules that are immuno-interactive with E-selectin, peptides thatbind to E-selectin and that block cell-cell adhesion, as well ascarbohydrate or peptide mimetics of E-selectin ligands. In someembodiments, the E-selectin antagonist reduces the expression of anE-selectin gene or the level or functional activity of an expressionproduct of that gene. For example, the E-selectin antagonist mayantagonize the function of E-selectin, including reducing or abrogatingthe activity of at least one of its ligand-binding sites. Alternatively,the E-selectin antagonist may act indirectly on E-selectin expression bymodulating the level or functional activity of a regulator ofE-selectin. For example, it is known that cytokine-dependent inductionof E-selectin expression is mediated through cooperative signalinginvolving the Ras/Raf protein kinase pathway and that inhibition ofC-rafantisense molecules can block E-selectin expression and E-selectinmediated cell-cell adhesion.

Illustrative agents for reducing or abrogating gene expression include,but are not restricted to, oligoribonucleotide sequences, includinganti-sense RNA and DNA molecules and ribozymes, that function to inhibitthe translation, for example, of E-selectin-encoding transcriptsincluding E-selectin mRNA. Representative transcripts of this typeinclude:

nucleotide sequences that comprise the sequence:

[SEQ ID NO: 1] atgattgcttcacagtttctctcagctctcactttggtgcttctcattaaagagagtggagcctggtcttacaacacctccacggaagctatgacttatgatgaggccagtgcttattgtcagcaaaggtacacacacctggttgcaattcaaaacaaagaagagattgagtacctaaactccatattgagctattcaccaagttattactggattggaatcagaaaagtcaacaatgtgtgggtctgggtaggaacccagaaacctctgacagaagaagccaagaactgggctccaggtgaacccaacaataggcaaaaagatgaggactgcgtggagatctacatcaagagagaaaaagatgtgggcatgtggaatgatgagaggtgcagcaagaagaagcttgccctatgctacacagctgcctgtaccaatacatcctgcagtggccacggtgaatgtgtagagaccatcaataattacacttgcaagtgtgaccctggcttcagtggactcaagtgtgagcaaattgtgaactgtacagccctggaatcccctgagcatggaagcctggtttgcagtcacccactgggaaacttcagctacaattcttcctgctctatcagctgtgataggggttacctgccaagcagcatggagaccatgcagtgtatgtcctctggagaatggagtgctcctattccagcctgcaatgtggttgagtgtgatgctgtgacaaatccagccaatgggttcgtggaatgtttccaaaaccctggaagcttcccatggaacacaacctgtacatttgactgtgaagaaggatttgaactaatgggagcccagagccttcagtgtacctcatctgggaattgggacaacgagaagccaacgtgtaaagctgtgacatgcagggccgtccgccagcctcagaatggctctgtgaggtgcagccattcccctgctggagagttcaccttcaaatcatcctgcaacttcacctgtgaggaaggcttcatgttgcagggaccagcccaggttgaatgcaccactcaagggcagtggacacagcaaatcccagtttgtgaagctttccagtgcacagccttgtccaaccccgagcgaggctacatgaattgtcttcctagtgcttctggcagtttccgttatgggtccagctgtgagttctcctgtgagcagggttttgtgttgaagggatccaaaaggctccaatgtggccccacaggggagtgggacaacgagaagcccacatgtgaagctgtgagatgcgatgctgtccaccagcccccgaagggtttggtgaggtgtgctcattcccctattggagaattcacctacaagtcctcttgtgccttcagctgtgaggagggatttgaattacatggatcaactcaacttgagtgcacatctcagggacaatggacagaagaggttccttcctgccaagtggtaaaatgttcaagcctggcagttccgggaaagatcaacatgagctgcagtggggagcccgtgtttggcactgtgtgcaagttcgcctgtcctgaaggatggacgctcaatggctctgcagctcggacatgtggagccacaggacactggtctggcctgctacctacctgtgaagctcccactgagtccaacattcccttggtagctggactttctgctgctggactctccctcctgacattagcaccatttctcctctggcttcggaaatgcttacggaaagcaaagaaatttgttcctgccagcagctgccaaagccttgaatcagatggaagctaccaaaagccttcttacatcctttaa;

nucleotide sequences that share at least 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99% sequence identity with SEQ ID NO: 1;

nucleotide sequences that hybridize under at least low, medium or highstringency conditions to SEQ ID NO: 1;

nucleotide sequences that encode the amino acid sequence:

[SEQ ID NO: 2] MIASQFLSALTLVLLIKESGAWSYNTSTEAMTYDEASAYCQQRYTHLVAIQNKEEIEYLNSILSYSPSYYWIGIRKVNNVWVWVGTQKPLTEEAKNWAPGEPNNRQKDEDCVEIYIKREKDVGMVNDERCSKKKLALCYTAACTNTSCSGHGECVETINNYTCKCDPGFSGLKCEQIVNCTALESPEHGSLVCSHPLGNFSYNSSCSISCDRGYLPSSMETMQCMSSGEWSAPIPACNVVECDAVTNPANGFVECFQNPGSFPWNTTCTFDCEEGFELMGAQSLQCTSSGNWDNEKPTCKAVTCRAVRQPQNGSVRCSHSPAGEFTFKSSCNFTCEEGFMLQGPAQVECTTQGQWTQQIPVCEAFQCTALSNPERGYMNCLPSASGSFRYGSSCFESCEQGFVLKGSKRLQCGPTGEWDNEKPTCEAVRCDAVHQPPKGLVRCAHSPIGEFTYKSSCAFSCEEGFELHGSTQLECTSQGQWTEEVPSCQVVKCSSLAVPGKINMSCSGEPVFGTVCKFACPEGWTLNGSAARTCGATGHWSGLLPTCEAPTESNIPLVAGLSAALGSLLTLAPFLLWLRKCLRKAKKFVPASSCQSLESD GSYQKPSYIL;

nucleotide sequences that encode an amino acid sequence that shares atleast 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequencesimilarity with SEQ ID NO: 2; and

nucleotide sequences that encode an amino acid sequence that shares atleast 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequenceidentity with SEQ ID NO: 2.

Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by binding to targeted mRNA and preventing protein translation.In regard to antisense DNA, oligodeoxyribonucleotides derived from thetranslation initiation site, e.g., between − and + regions arepreferred.

In some embodiments, anti-sense RNA and DNA molecules are used todirectly block the translation of E-selectin mRNA by binding to targetedmRNA and preventing protein translation. In regard to antisense DNA,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between −10 and +10 regions are desirable. Illustrative E-selectinantisense molecules are described, for example, by Bennett et al. (1994,J. Immunol., 152(7): 3530-3540) and by Baker el al. (U.S. Pat. No.5,789,573). In other embodiments, C-rafantisense molecules can be used,which block C-raf expression, leading to reduced or abrogated E-selectinexpression, as disclosed for example by Khatib et al. (2002, Cancer Res.62(19):5393-5398).

In other embodiments, anti-E-selectin ribozymes are used for catalyzingthe specific cleavage of E-selectin RNA. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by a endonucleolytic cleavage.Within the scope of the invention are engineered hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of target sequences. Specific ribozyme cleavagesites within any potential RNA target are initially identified byscanning the target molecule for ribozyme cleavage sites which includethe following sequences, GUA, GUU and GUC. Once identified, short RNAsequences of between 15 and 20 ribonucleotides corresponding to theregion of the target gene containing the cleavage site may be evaluatedfor predicted structural features such as secondary structure that mayrender the oligonucleotide sequence unsuitable. The suitability ofcandidate targets may also be evaluated by testing their accessibilityto hybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

Both anti-sense RNA and DNA molecules and ribozymes may be prepared byany method known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligodeoxyribonucleotideswell known in the art such as for example solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding the antisenseRNA molecule. Such DNA sequences may be incorporated into a wide varietyof vectors which incorporate suitable RNA polymerase promoters such asthe T7 or SP6 polymerase promoters. Alternatively, antisense cDNAconstructs that synthesize antisense RNA constitutively or inducibly,depending on the promoter used, can be introduced stably into celllines.

Various modifications to the DNA molecules may be introduced as a meansof increasing intracellular stability and half-life. Possiblemodifications include but are not limited to the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

In other embodiments, RNA molecules that mediate RNA interference (RNAi)of an E-selectin gene or E-selectin transcript can be used to reduce orabrogate gene expression. RNAi refers to interference with ordestruction of the product of a target gene by introducing a singlestranded, and typically a double stranded RNA (dsRNA) that is homologousto the transcript of a target gene. Thus, in some embodiments, dsRNA perse and especially dsRNA-producing constructs corresponding to at least aportion of an E-selectin gene may be used to reduce or abrogate itsexpression. RNAi-mediated inhibition of gene expression may beaccomplished using any of the techniques reported in the art, forinstance by transfecting a nucleic acid construct encoding a stem-loopor hairpin RNA structure into the genome of the target cell, or byexpressing a transfected nucleic acid construct having homology for anE-selectin gene from between convergent promoters, or as a head to heador tail to tail duplication from behind a single promoter. Any similarconstruct may be used so long as it produces a single RNA having theability to fold back on itself and produce a dsRNA, or so long as itproduces two separate RNA transcripts which then anneal to form a dsRNAhaving homology to a target gene.

Absolute homology is not required for RNAi, with a lower threshold beingdescribed at about 85% homology for a dsRNA of about 200 base pairs(Plasterk and Ketting, 2000, Current Opinion in Genetics and Dev. 10:562-67). Therefore, depending on the length of the dsRNA, theRNAi-encoding nucleic acids can vary in the level of homology theycontain toward the target gene transcript, i.e., with dsRNAs of 100 to200 base pairs having at least about 85% homology with the target gene,and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about75% homology to the target gene. RNA-encoding constructs that express asingle RNA transcript designed to anneal to a separately expressed RNA,or single constructs expressing separate transcripts from convergentpromoters, are suitably at least about 100 nucleotides in length.RNA-encoding constructs that express a single RNA designed to form adsRNA via internal folding are usually at least about 200 nucleotides inlength.

The promoter used to express the dsRNA-forming construct may be any typeof promoter if the resulting dsRNA is specific for a gene product in thecell lineage targeted for destruction. Alternatively, the promoter maybe lineage specific in that it is only expressed in cells of aparticular development lineage. This might be advantageous where someoverlap in homology is observed with a gene that is expressed in anon-targeted cell lineage. The promoter may also be inducible byexternally controlled factors, or by intracellular environmentalfactors.

In other embodiments, RNA molecules of about 21 to about 23 nucleotides,which direct cleavage of specific mRNA to which they correspond, as forexample described by Tuschl et al. in U.S. Patent ApplicationPublication No. 20020086356, can be utilized for mediating RNAi. Such21-23 nt RNA molecules can comprise a 3′ hydroxyl group, can besingle-stranded or double stranded (as two 21-23 nt RNAs) wherein thedsRNA molecules can be blunt ended or comprise overhanging ends (e.g.,5′, 3′).

Illustrative RNAi molecules are commercially available from Santa CruzBiotechnology, Inc. (Santa Cruz, Calif., USA).

In still other embodiments, the functional activity of an E-selectinpolypeptide in the cell is inhibited through use of an anti-E-selectinantigen-binding molecule (e.g., a neutralizing antibody) as describedfor example by Owens et al. in U.S. Pat. No. 6,407,214. A range ofanti-E-selectin antibodies are available commercially, for example fromAbcam (Cambridge, UK), Beckman Coulter (Fullerton, Calif., USA), BenderMedSystems (Vienna, Austria), BioGenex (San Ramon, Calif., USA), BiomedaCorporation (Foster City, Calif., USA), BioVision, Inc. (Mountain View,Calif., USA), Cell Sciences (Canton, Mass., USA), Covance ResearchProducts (Denver, Pa., USA), Celltech Chiroscience (Slough, UK; CDP850humanized monoclonal antibody against E-selectin), GeneTex (San Antonio,Tex. USA), Hycult Biotechnology BV (Uden, The Netherlands), ProteinDesign Labs (Freemont, Calif., USA; SMART HuEP57C humanized monoclonalantibody), and R&D Systems (Minneapolis, Minn., USA).

In some embodiments, the E-selectin antagonist is selected from peptideinhibitors of E-selectin. Representative inhibitors of this type aredisclosed, for example, by Cwirla et al. in International Publication WO94/25043, which is expressly incorporated herein by reference in itsentirety, and include peptides of from 9 to 20 amino acids having a corestructure comprising:

WXXLWXXX′ [SEQ ID NO: 3] where each amino acid is indicated by theone-letter amino acid code where specifically W is tryptophan, L isleucine, and X is any amino acid and X′ is selected from the groupconsisting of M and Nle where M is methionine and Nle is norleucine.Specific peptides having this core structure comprise the sequenceX,X₂X₃WX₄X₅LWX₆X₇X₈X₉ [SEQ ID NO: 4], wherein each residue can beindependently selected as follows: X is H, E, or D; X₂ is I, M, or Nle;X₃ is T or S; X₄ is D, E, or L; X₅ is Q or E; X* is N or D; X₇ is L, M,V, or I; X₈ is M or Nle; and X, is N, S, Q.

In specific embodiments, the peptides are selected from the followingsequences:

[SEQ ID NO: 5] DGDITWDQLWDLMK; [SEQ ID NO: 6] DYTWFELWDMMQ;[SEQ ID NO: 7] DITWDELWKIMN; [SEQ ID NO: 8] QITWAQLWNMMK; [SEQ ID NO: 9]DYSWHDLWEMMS; [SEQ ID NO: 10] DITWDQLWDLNleK; [SEQ ID NO: 11]HITWDQLWRIMT; [SEQ ID NO: 225] d-DITWDQLWDLMK;  [SEQ ID NO: 226]Dd-ITWDQLWDLMK;  [SEQ ID NO: 227] DId-TWDQLWDLMK; [SEQ ID NO: 228]DITWd-DQLWDLMK;  [SEQ ID NO: 229] DITWDQLWDd-LMKU;  [SEQ ID NO: 230]DITWDQLWDLMd-K;  and  [SEQ ID NO: 12] HITWDQLWNVMN; [SEQ ID NO: 231]ITWDQLWDLMK  (amino acids 4-14 of SEQ ID NO: 5); [SEQ ID NO: 232]DITWDQLWDLMK  acids 3-14 of SEQ ID NO: 5); [SEQ ID NO: 13]DGDFTWDQLWDLMK; [SEQ ID NO: 14] DYTWFELWDMMQ; [SEQ ID NO: 15]DITWDELWKIMN; [SEQ ID NO: 16] QITWAQLWNMMK; [SEQ ID NO: 17]DYSWHDLWEMMS; [SEQ ID NO: 18] DITWDQLWDLNleK; [SEQ ID NO: 19]ATTWDQLWLLMS; [SEQ ID NO: 20] ELTWDQLWVLMS; [SEQ ID NO: 21]DVTWDQLWELMT; [SEQ ID NO: 22] EVTWDQLWVMMQ; [SEQ ID NO: 23]NLTWDQLWVLMS; [SEQ ID NO: 24] EMSWLELWNVMN; [SEQ ID NO: 25]TITWDQLWQMMS; [SEQ ID NO: 26] ELSWDQLWNVMN; [SEQ ID NO: 27]EMTWQELWNVMN; [SEQ ID NO: 28] EMTWTELWNVMN; [SEQ ID NO: 29]DMTWSQLWNVMN; [SEQ ID NO: 30] EMTWLGLWNVMN; [SEQ ID NO: 31]QITWMELWNLMN; [SEQ ID NO: 32] ETTWDQLWEVMN; [SEQ ID NO: 33]ETTWDQLWDVMN; [SEQ ID NO: 34] DISWDQLWNVMN; [SEQ ID NO: 35]QITWDQLWDLMK; [SEQ ID NO: 36] EMTWDQUWNVMN; [SEQ ID NO: 37]DITWDQUWNMMD; [SEQ ID NO: 38] DITWNMLWNMMQ; [SEQ ID NO: 39]DISWDDLWIMMN; [SEQ ID NO: 40] DITWHQLWNLMN; [SEQ ID NO: 41]EISWEQLWFMMN; [SEQ ID NO: 42] DITWEQLWNMMN; SEQ ID NO: 43] EITWDQLWTLMT;[SEQ ID NO: 44] DITWHQLWNLMN; [SEQ ID NO: 45] DMTWDQLWIVMN;[SEQ ID NO: 46] DITWEQLWNLMN; [SEQ ID NO: 47] QITWYQLWNMMN;[SEQ ID NO: 48] HISWHELWNLMQ; [SEQ ID NO: 49] YTTWEQLWTMMN;[SEQ ID NO: 50] HITWDQLWDLMQ; [SEQ ID NO: 51] QITWDQLWDLMY;[SEQ ID NO: 52] QITVDQLWNMMI; [SEQ ID NO: 53] YITWEQLWNMMN;[SEQ ID NO: 54] HITWDQLWDFMS; [SEQ ID NO: 55] HITWDQLWEIMS;[SEQ ID NO: 56] HITWDQLWALMT; [SEQ ID NO: 57] HITWDQLWSLMS;[SEQ ID NO: 58] HITWDQLWLMMS; [SEQ ID NO: 59] HITWDQLWDLMQ;[SEQ ID NO: 60] HITWDQLWWFMA; [SEQ ID NO: 61] HITWDQLWLLMA;[SEQ ID NO: 62] HITWDQUWWILMA; [SEQ ID NO: 63] GSDSHTTWDELWNLMNPVLA;[SEQ ID NO: 64] NWLDDITWDELWIUMNPSTA; [SEQ ID NO: 65]ETDDHITWDQLWRFMTATMA; [SEQ ID NO: 66] WTDTHITWDQLWHFMNMGEQ;[SEQ ID NO: 67] GFGEAITWDQLWDMMNGEDA; [SEQ ID NO: 68]NVAEQITWDQLWNLMSVGSS; [SEQ ID NO: 69] GQTGLITWDMLWNLMNPVGE;[SEQ ID NO: 70] GTGDHITWDQLWNLMINEKG; [SEQ ID NO: 71]EYGRHITWDQLWQLMQSATA; [SEQ ID NO: 72] MNNWHVSWEQLWDIMNGPPN;[SEQ ID NO: 73] ESASHITWGQLWDLMNASEV; [SEQ ID NO: 74]YWRGNITWDQLWNIMNSEYS; [SEQ ID NO: 75] AGASHITWAQLWNMMNGNEG;[SEQ ID NO: 76] GSWAHITWDQLWNLMNMGIQ; [SEQ ID NO: 77]YGNSNITWDQLWSFMNRQTT; [SEQ ID NO: 78] AHLPH1SWDTLWHIMNKGEK;[SEQ ID NO: 79] ESASHITWGQLWDLMNASEV; [SEQ ID NO: 80]MNNWHVSWEQLWDIMNGPPN; [SEQ ID NO: 81] GFGEAITWDQLWDMMNGEDA;[SEQ ID NO: 82] WTDTHITWDQLWHFMNMGEQ; [SEQ ID NO: 83] EMTWAELMTLME;[SEQ ID NO: 84] DISWRQLWDIMN; [SEQ ID NO: 85] EISWLGLWDIMN;[SEQ ID NO: 86] DMTWHDLWTLMS; [SEQ ID NO: 87] RGVWGGLWSMTW;[SEQ ID NO: 88] EMTWQQLWWMQ; [SEQ ID NO: 89] AEWFWDQLWHVMNPAESQ;[SEQ ID NO: 90] RNMSWLELWEHMK; [SEQ ID NO: 91] SQVTWNDLWSVMNPEVVN;[SEQ ID NO: 92] FIRAEWLALWEQMSP; [SEQ ID NO: 93] YKKEWLELWHQMQA;[SEQ ID NO: 94] RSLSWLQLWDQMK; [SEQ ID NO: 95] KEQQWRNLWKMMS;[SEQ ID NO: 96] KKEDWLALWRIMSVPD; [SEQ ID NO: 97] RNMSWLELWEHMK;[SEQ ID NO: 98] GRPTWNELWDMMQAP; [SEQ ID NO: 99] KRKQWIELWNIMS;[SEQ ID NO: 100] KTSEWNNLWKLMSQ  [SEQ ID NO: 101] HVSWEQLWDIMN [SEQ ID NO: 102] KKEDWLALWRIMSV; [SEQ ID NO: 103] HRAEWLALWEQMS;[SEQ ID NO: 104] DGDITWDQLWDLNleK; [SEQ ID NO: 105] QITWDQLWDLNleK;[SEQ ID NO: 106] AETWDQLWHVMNPAESQ; [SEQ ID NO: 107] DITWAQLWNNleNleN; and

DITWDQLWDLM [SEQ ID NO: 233](amino acids 3-13 of SEQ ID NO: 5);DITWDQLWDL [SEQ ID NO: 234](amino acids 3-12 of SEQ ID NO: 5);TWDQLWDLMK [SEQ ID NO: 235](amino acids 5-14 of SEQ ID NO: 5); andDITWDQLWDLMK-C(O)NH₂ [SEQ ID NO: 108](amino acids 3-14 of SEQ ID NO: 5)wherein d-indicates a D-amino acid and —C(O)NH₂ represents an amidatedcarboxy terminus.

Alternative peptide inhibitors of E-selectin can be selected from thosedisclosed by Barrett et al. in International Publication WO 95/31210,which is expressly incorporated herein by reference in its entirety, andwhich include peptides and peptide mimetics comprising: a molecularweight of less than about 2000 daltons, and a binding affinity toE-selectin as expressed by an IC₅₀-HL6O of no more than about 100 μMwherein from zero to all of the —C(O)NH— linkages of the peptide havebeen replaced by a linkage selected from the group consisting of a—CH₂OC(O)NR— linkage, a phosphonate linkage, a —CH₂S(O)₂N— linkage, a—CH₂NR— linkage, a —C(O)NR⁶— linkage, and a —NHC(O)NH— linkage where Ris hydrogen or lower alkyl, and R⁶ is lower alkyl, further wherein theN-terminus of said peptide or peptide mimetic is selected from the groupconsisting of a —NRR¹ group, a —NRC(O)R group, a —NRC(O)OR group, a—NRS(O)₂R group, a —NHC(O)NHR group, a succinimide group, abenzyloxycarbonyl-NH— group, and a benzyloxycarbonyl-NH— group havingfrom 1 to 3 substituents on the phenyl ring selected from the groupconsisting of lower alkyl, lower alkoxy, chloro, and bromo, where R andR¹ are independently selected from the group consisting of hydrogen andlower alkyl, and still further wherein the C-terminus of said peptide orpeptide mimetic has the formula —C(O)R² where R² is selected from thegroup consisting of hydroxy, lower alkoxy, and —NR³R⁴ where R³ and R⁴are independently selected from the group consisting of hydrogen andlower alkyl and where the nitrogen atom of the —NR³R⁴ group canoptionally be the amine group of the N-terminus of the peptide so as toform a cyclic peptide and physiologically acceptable salts thereof

Representative peptides and peptide mimetics disclosed in WO 95/31210include peptides which comprise the following sequences:

[SEQ ID NO: 109] YDDVCCELLF; [SEQ ID NO: 110] DLPQWYTEWC;[SEQ ID NO: 111] ENSHWCTCPC; [SEQ ID NO: 112] DIEQDWVTWM;[SEQ ID NO: 113] NEWCWPCRL; [SEQ ID NO: 114] DIWQDWVRWM;[SEQ ID NO: 115] DLWQDWVTWM; [SEQ ID NO: 116] DLWQDWVHWM;[SEQ ID NO: 117] DIWQDWVTWM; [SEQ ID NO: 118] DIWQDWVKWM [SEQ ID NO: 119] DIWQDWVRWM-C(O)NH₂; [SEQ ID NO: 120] DIWEDWVRWM;[SEQ ID NO: 121] DIWQDWFFWM; [SEQ ID NO: 122] DITNal(1)DQLWDLMK-C(O)NH₂;[SEQ ID NO: 123] DITWIDQLNal(1)DLMK-C(O)NH₂; [SEQ ID NO: 124]DITNal(2)DQLWDLMK-C(O)NH₂; [SEQ ID NO: 125] DITWDQLNal(2)DLMK-C(O)NH₂;[SEQ ID NO: 126] DITChaDQLWDLMK-C(O)NH₂; [SEQ ID NO: 127]DITWDQLChaDLMK-C(O)NH₂; [SEQ ID NO: 128] DITWDQLWDLM(OCH₃)K-C(O)NH₂;[SEQ ID NO: 129] DITWDQLWDLM(SOCH₃)K-C(O)NH₂; [SEQ ID NO: 130]DITWDQLWDLM(SOCH₃)K-C(O)NH₂; [SEQ ID NO: 131] DITWDQLW-Aib-LMK-C(O)NH₂;[SEQ ID NO: 132] DITWDQLW-Aib-LMK; [SEQ ID NO: 133] DITW-Aib-QLWKLMK;[SEQ ID NO: 134] DITW-Alb-QLWDLMK-C(O)NH₂; [SEQ ID NO: 135]DITW-Alb-QLW-Aib-LMK-CONH₂; [SEQ ID NO: 136] DITW-Aib-QLWDLMK;[SEQ ID NO: 137] DITW-Aib-QLW-Aib-LMK; [SEQ ID NO: 138] AITWDQLWDLNleK;[SEQ ID NO: 139] DATWDQLWDLNleK; [SEQ ID NO: 140] DITADQLWDLNleK;[SEQ ID NO: 141] DITWAQLWDLNleK; [SEQ ID NO: 142] DITWDALWDLNleK;[SEQ ID NO: 143] DITWDQAWDLNleK; [SEQ ID NO: 144] DITWDQLADLNleK;[SEQ ID NO: 145] DITWDQLWALNleK; [SEQ ID NO: 146] DITWDQLWDANleK;[SEQ ID NO: 147] DITWDQLWDLAK; [SEQ ID NO: 148] DITWDQLWDLNleA;[SEQ ID NO: 149] DITNal(1)DQLNal(1)DLMK-C(O)NH₂; [SEQ ID NO: 150]DITWAQL.Nal(l)DLN4K-C(O)NH₂; [SEQ ID NO: 151]DITNal(1)AQLNal(1)DLMK-C(O)NH₂; [SEQ ID NO: 152]DITNal(1)AQLNal(1)DLM(OCH₃)K-C(O)NH₂; [SEQ ID NO: 153]DITWAQLWDLM(SO₂CH₃)K-C(O)NH₂; [SEQ ID NO: 154]DITWAQLNal(1)DLM(SO₂CH₃)K-C(O)NH₂; [SEQ ID NO: 155]DITNal(1)AQLWDLM(SO₂CH₃)K*C(O)NH₂; [SEQ ID NO: 156]DITWAQLWDLM(OCH₃)K-(O)NH₂; [SEQ ID NO: 157] DITNal(1)AQLWDLMK-C(O)NH₂;[SEQ ID NO: 158] DITNal(1)DQLWDLM(SO₂CH₃)K-C(O)NH₂; [SEQ ID NO: 159]DITWDQLNal(1)DLM(SO₂CH₃)K-C(O)NH₂; [SEQ ID NO: 160]DITWAQLWDLMK-C(O)NH₂; [SEQ ID NO: 161] DITWAHQLWDLMK-C(O)NH₂;[SEQ ID NO: 162] Ac-DITWDQLWKLMK  [SEQ ID NO: 163]Ac-DITWDQLWDL-Nle-K-C(O)NH₂; [SEQ ID NO: 164] Succ-ITWDQLWDLMK;[SEQ ID NO: 165] Cbz-TWDQLWDLMK; [SEQ ID NO: 166]Succ-ITWDQUWDLMK-C(O)NH₂; [SEQ ID NO: 167] Cbz-DITWDQLWDLMK-C(O)NH₂;[SEQ ID NO: 168] Ac-DITWDQLWDLMK-C(O)NH₂; [SEQ ID NO: 169]Cbz-ITWDQLWDLNIK; [SEQ ID NO: 170] Succ-ITWDQLWAibLM(SO₂CH₃)K-C(O)NH₂;[SEQ ID NO: 171] Succ-ITWAQLWAibLM(SO₂CH₃)K-C(O)NH₂; [SEQ ID NO: 172]Succ-ITWAQLWDLM(SO₂CH₃)K-C(O)NH₂; [SEQ ID NO: 173]Succ-ITWAQLWDLM(OCH₃)K-C(O)NH₂; [SEQ ID NO: 174]Succ-ITWAQLWAibLM(OCH)K-C(O)NH₂; [SEQ ID NO: 175]Succ-ITWDQLWAibLM(SOCH₃)K-C(O)NH₂; [SEQ ID NO: 176]Ac-DITWAQLWDLMK-C(O)NH₂; [SEQ ID NO: 177]Ac-DITWDQLWAibILM(OCH₃)K-C(O)NH₂; [SEQ ID NO: 178]Ac-DITWAQLWAibLM(SO₂(14₃)K-C(O)NH₂; [SEQ ID NO: 179]Ac-DITWDQLWAibLM(S0₂CH₃)K-C(O)NH₂; [SEQ ID NO: 180]Ac-DMVAQLWDLM(SO₂CH₃)K-C(O)NH₂; [SEQ ID NO: 181]Ac-DMVAQLWDLM(OCH₃)K-C(O)NH₂; [SEQ ID NO: 182]Ac-DITWAQLWAibLM(OCH₃)K-C(O)NH₂; [SEQ ID NO: 183]Ac-DITWDQLWAibLM(OCH₃)K-C(O)NH₂; [SEQ ID NO: 184]DITWAQLWAibLM(OCH₃)K-C(O)NH₂; [SEQ ID NO: 185]DITWAQLWAibLM(SO₂CH₃)K-C(O)NH₂; [SEQ ID NO: 186]DITWDQUWAibLM(OCH₃)K-C(O)NH₂; [SEQ ID NO: 187] WDLMK-C(O)NH₂;[SEQ ID NO: 188] Cbz-WDLM-C(O)NH₂; [SEQ ID NO: 189] Chz-QLWD-C(O)NH₂;[SEQ ID NO: 190] Chz-QLWDLM-C(O)NH₂; [SEQ ID NO: 191] Cbz-ITWDQ-C(O)NH₂;[SEQ ID NO: 192] Cbz-TWDQLW-C(O)NH₂; [SEQ ID NO: 193] Cbz-WDQLWD-CONH₂;[SEQ ID NO: 194] Cbz-ITWAQ-C(O)NH_(2;) [SEQ ID NO: 195]Cbz-ITWDQL-C(O)NH₂; N-Cbz, N-Me-ITW-C(O)NH₂; Cbz-IT-[(Me)(DL)W]-C(O)NH₂;[SEQ ID NO: 196] N-Cbz, N-Me-ITWDQ-C(O)NH₂; [SEQ ID NO: 197]Cbz-ITW-N-Me-DQ-C(O)NH₂; [SEQ ID NO: 198] Cbz-117-N-111e-WDQ-C(O)NH₂;[SEQ ID NO: 199] Chz-I-(N-Me T)WDQ-C(O)NH₂; Chz-I-(N-Me-T)W-C(O)NH₂;[SEQ ID NO: 200] Cbz-IT-RaMe)(DL)Wl-DQ-C(O)NH₂;Cbz-N-Me-I-T-[(aMe)(DL)W]-C(O)NH₂; [SEQ ID NO: 201] DITWDELWTLML;[SEQ ID NO: 202] HLTWDQLWRIMN; [SEQ ID NO: 203] HITWDQLWNLMN;[SEQ ID NO: 204] HITEDQLWDFMN; [SEQ ID NO: 205] HVTWELLWDIMN;[SEQ ID NO: 206] HITWGQLWDLMN; [SEQ ID NO: 207] HITWEQLWDLMN;[SEQ ID NO: 208] EITWFELWEWME; [SEQ ID NO: 209] MASWVLLWPYMG-C(O)NH₂;[SEQ ID NO: 210] DITWAQLWNIMN;

where Aib is aminoisobutryic acid, Nal(1) is α-naphthylalanine, Nal(2)is β-naphthylalanine, M(SO₂CH₃) is methionine sulfone, M(OCH₃) isO-methylmethione, Cbz is benzoxycarbonyl, Ac is acetyl, Succ issuccinimidyl, and N-Me is a methylated nitrogen on the amine or amidegroup as designated therein.

Alternative peptide inhibitors of E-selectin are disclosed for examplein US Pat. Appl. Pub. No 2005/0181987, which is expressly incorporatedherein by reference in its entirety, and which discloses severalpeptido-mimetics which mimic the topography of the E-selectin ligand:ASAVNLYIPTQE [SEQ ID NO: 211], VYLAPGRISRDY [SEQ ID NO: 212],VYLAPGRFSRDY [SEQ ID NO: 213], CTSHWGVLSQRR [SEQ ID NO: 214].RVLSPESYLGPS [SEQ ID NO: 215], RVLSPESYLGPA [SEQ ID NO: 216],VGNGVLMGRRG [SEQ ID NO: 217], RVLSPESYLGPA [SEQ ID NO: 218],GNCRYIGLRQFG [SEQ ID NO: 219], DIRVEPGGGYTH [SEQ ID NO: 220],APIHTYTGRARG [SEQ ID NO: 221], and RHTCVRSCGHDR [SEQ ID NO: 222].

In other embodiments, the peptide inhibitors are glycopeptide molecules.Representative molecules of this type are disclosed, for example, byCummings et al. in International Publication WO 99/065712, which isexpressly incorporated herein by reference in its entirety. Inparticular, this reference discloses glycosulfopeptides (GSPs) whichhave one or more sulfated tyrosine residues and a glycan linked to thepeptide, the glycan desirably including a sialyl Lewis^(x) group or asialyl Lewis^(a) group. Illustrative GSPs of this type have an O-glycancomprising a β1,6 linkage to a GalNAc. Several exemplary GSPs aredisclosed including compounds represented by the formula:

wherein: Tyr is a tyrosine residue; SO₃ is a sulfate group attached tothe tyrosine residue; X_(A) is an N- or O-linking amino acid residue; Ris a sialylated, fucosylated, N-acetyllactosamino glycan in O- orN-linkage to X_(A); X_(B), X_(C), and X_(D) are amino acid residues; andj, k and n are each from 0 to 12, wherein each amino acid sequence[X_(B)]j [X_(C)]_(k), or [X_(D)]_(n) comprises from 0 to 12 amino acidresidues. In illustrative examples of this type, the compound comprisesno more than 38 amino acids.

In specific embodiments, X comprises one or two sulfated tyrosineresidues; j=0 to 10, k=0 to 5, and n=0 to 10; R is selected from thegroup consisting of R₁-R₁₅; j=0, k=0 to 5 and n=0; X_(B) comprisesproline; X_(C) comprises tyrosine; the compound further comprises atleast one additional sialylated, fucosylated O-glycan linked to an aminoacid residue; X_(A) is an O-linking amino acid; the O-linking amino acidresidue is serine or threonine; X_(A) is an N-linking amino acid; Rcomprises a β1,6 linkage to a GAlNAc; and/or R is core-2 based.

In other embodiments, suitable GSPs are selected from the compoundsdisclosed by Cummings el al in International Publication No. WO2003/032925, which is expressly incorporated herein by reference in itsentirety. Representative GSPs disclosed in the reference have theformula:

wherein: Tyr is a tyrosine residue; C is an N-, S-, or O-linking aminoacid residue; R is a sialylated, fucosylated, N-acetyllactosaminoglycanin O-, S-, or N-linkage to C; A, B, and D are amino acid sequences eachcomprising from 0 to 12 amino acid residues. In specific embodiments Cis serine, threonine, hydroxyproline, tyrosine, lysine, hydroxylysine,methionine, cysteine, asparagine or glutamine; the glycosulfopeptide isconjugated, linked or complexed to a polymeric carrier molecule (e.g.,PEG); A of the glycosulfopeptide comprises X₁-X₂-X₃-X₄-X₅, wherein X₁and X₃ are sulfated tyrosines and X₂, X₄ and X₅ are amino acids selectedfrom the group consisting of Ala, Asp, Cys, Glu, Phe, Gly, His, lie,Lys, Leu, Met, Asn, Pro, Gly, Org, Ser, Thr, Val, Trp, and Tyr, or isabsent; B of the glycosulfopeptide is X₆-X₇-X₈-X₉-X₁₀ wherein each ofX₆-X₁₀ is an amino acid selected from the group consisting of Ala, Asp,Cys, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gly, Org, Ser,Thr, Val, Trp, and Tyr, or is absent; D of the glycosulfopeptide isX₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆ wherein each of X₁₁-X₁₆ is an amino acidselected from the group consisting of Ala, Asp, Cys, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gly, Org, Ser, Thr, Val, Trp, and Tyr, oris absent

In still other embodiments, the peptide inhibitor of E-selectin isAc-TWDQLWDLMK-CONH₂ as disclosed for example by Rinnbauer et al. (2003,Glycobiology 13(6), 435-443), which is expressly incorporated herein byreference in its entirety.

In still other embodiments, the E-selectin antagonist is selected fromcarbohydrate inhibitors of E-selectin. In illustrative examples of thistype, the carbohydrate inhibitor is selected from the compoundsdescribed by Wong et al. in U.S. Pat. No. 5,830,871, which is expresslyincorporated herein by reference in its entirety. In some embodiments,these compounds are represented by any one of the following formulae:

In the above formulas, R₁ is a radical selected from the groupconsisting of —H, —OH, —O—C₁-C₆, —OBn, —N₃, —OSO₃ ²⁻,—OCOCH₂CH₂CONHCH(CH₂CO₂H)CO₂H, and —NHR′. R′ is a radical selected fromthe group consisting of alkyl (C₁-C₆), acyl, decanoyl, phenylacetyl, and—COCH₂CH₂CO₂H. R₂ is a radical selected from the group consisting of—CH₂PO₃ ²⁻ and —OPO₃ ²⁻.

Other embodiments of the carbohydrate inhibitors disclosed by Wong etal., are represented by the following formulae:

In these embodiments, R₁ is a radical selected from the group consistingof —CH₂POPO₃ ²⁻ and —OPOPO₃ ²⁻.

Still other embodiments of the carbohydrate inhibitors disclosed by Wonget al., are represented by the following formulae:

In the above formulas: R₁ is a radical selected from the groupconsisting of —H, —OH, —O-alkyl (C₁-C₆), —OBn, —N₃, —OPOPO₃ ²⁻,—OCOCH₂CH₂CONHCH(CH₂CO₂H) CO₂H, and —NHR′; R′ is a radical selected fromthe group consisting of alkyl (C₁-C₆), acyl, decanoyl, phenylacetyl, and—COCH₂CH₂CO₂H; R₂ is a radical selected from the group consisting of—CH₂PO₃ ²⁻ and OPO₃ ²⁻; and “n” runs from 1 to 4.

In some embodiments, the carbohydrate inhibitor is a derivative ofsialyl-Lewis^(x) (SLe^(x)) or sialyl-Lewis^(a) (SLe^(a)), illustrativeexamples of which are described by Thoma et al. in InternationalPublication No. WO 98/06730, which is expressly incorporated herein byreference in its entirety.

In other illustrative examples, the carbohydrate inhibitor is selectedfrom the oligosaccharide or glycomimetic compounds described by Magnaniet al. in International Publication Nos. WO 2008/100453 and WO2008/060378, which are expressly incorporated herein by reference intheir entirety. These compounds are represented by the formula:

wherein:

R¹═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, halogenated C₁-C₈alkanyl, aryl or heteroaryl either of which may be substituted with oneor more of Me. OMe, halide, OH, or NHX where X═H, C₁-C₈ alkanyl, C₁-C₈alkenyl, C₁-C₈ alkynyl, halogenated C₁-C₈ alkanyl, aryl or heteroaryleither of which may be substituted with one or more of Me, OMe, halide,or OH; C(═O)OX, alkanyl substituted with C(═O)OX, C(═O)NHX, alkanylsubstituted with C(═O)NHX, where X═C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈alkynyl, halogenated C₁-C₈ alkanyl, aryl or heteroaryl either of whichmay be substituted with one or more of Me, OMe, halide, or OH; O(═O)X,OX, NHX, NH(═O)X, where X═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈alkynyl, halogenated C₁-C₈ alkanyl, aryl or heteroaryl either of whichmay be substituted with one or more of Me, OMe, halide, or OH;

R²═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, halogenated C₁-C₈alkanyl, aryl or heteroaryl either of which may be substituted with oneor more of Me, OMe, halide, OH₁ or NHX where X═H₁ C₁-C₈ alkanyl, C₁-C₈alkenyl, C₁-C₈ alkynyl, halogenated C₁-C₈ alkanyl, aryl or heteroaryleither of which may be substituted with one or more of Me, OMe, halide,or OH; —C(═O)OX where X is C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,aryl or heteroaryl either of which may be substituted with one or moreof Me, OMe, halide, or OH; —C(═O)NH(CH₂)_(n)NH₂ where n=0-30, C(═O)NHXor CX₂OH, where X═C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,halogenated C₁-C₈ alkanyl, aryl or heteroaryl either of which may besubstituted with one or more of Me, OMe, halide, or OH; O(═O)X, OX. NHX,NH(═O)X, where X═H₁ C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,halogenated C₁-C₈ alkanyl, aryl or heteroaryl either of which may besubstituted with one or more of Me, OMe, halide, or OH; with the provisothat R¹ and R² are not both H;

the cyclohexane derivative is at least attached to the oligosaccharideor glycomimetic compound at an OH, R¹ or R².

In some embodiments, the oligosaccharide or glycomimetic compoundscomprise:

wherein:

R¹═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, halogenated C₁-C₈alkanyl, aryl or heteroaryl either of which may be substituted with oneor more of Me, OMe, halide, OH, or NHX where X═H₁ C₁-C₈ alkanyl, C₁-C₈alkenyl, C₁-C₈ alkynyl, halogenated C₁-C₈ alkanyl, aryl or heteroaryleither of which may be substituted with one or more of Me, OMe₁ halide,or OH; C(═O)OX, alkanyl substituted with C(═O)OX, C(═O)NHX, alkanylsubstituted with C(═O)NHX, where X═C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈alkynyl, halogenated C₁-C₈ alkanyl, aryl or heteroaryl either of whichmay be substituted with one or more of Me, OMe, halide, or OH; O(═O)X,OX, NHX, NH(═O)X, where X═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈alkynyl, halogenated C₁-C₈ alkanyl, aryl or heteroaryl either of whichmay be substituted with one or more of Me, OMe, halide, or OH;

R²═H₁ C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, halogenated C₁-C₈alkanyl, aryl or heteroaryl either of which may be substituted with oneor more of Me, OMe, halide, OH, or NHX where X═H, C₁-C₈ alkanyl, C₁-C₈alkenyl, C₁-C₈ alkynyl, halogenated C₁-C₈ alkanyl, aryl or heteroaryleither of which may be substituted with one or more of Me, OMe, halide,or OH; —C(═O)OX where X is C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,aryl or heteroaryl either of which may be substituted with one or moreof Me, OMe, halide, or OH; —C(═O)NH(CH₂)nNH₂ where n=0-30, C(═O)NHX orCX₂OH, where X═C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, halogenatedC₁-C₈ alkanyl, aryl or heteroaryl either of which may be substitutedwith one or more of Me, OMe, halide, or OH; O(═O)X, OX. NHX₁ NH(═O)X,where X═H₁ C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, halogenatedC₁-C₈ alkanyl, aryl or heteroaryl either of which may be substitutedwith one or more of Me, OMe, halide, or OH; with the proviso that R¹ andR² are not both H;

—O—C(═O)—X, —NH₂, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2 and X isindependently selected from C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,

where Q is H or a physiologically acceptable salt, C₁-C₈ alkanyl, CrC₈alkenyl, CrC₈ alkynyl, aryl, heteroaryl, (CH₂)m-aryl or(CH₂)m-heteroaryl where m is 1-10, and where n=0-10, and any of theabove ring compounds may be substituted with one to three independentlyselected of Cl, F, CF₃, CrC₈ alkoxy, NO₂, C₁-C₈ alkanyl, C₁-C₈ alkenyl,C₁-C₈ alkynyl, C₁-C₁₄ aryl, or OY, C(═O)OY, NY₂ or C(═O)NHY where Y isH, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, or C₁-C₁₄ aryl;

6′sulfated GIcNAc, 6′carboxylated GIcNAc, 6′sulfated GaINAc, 6′sulfatedgalactose, 6′carboxylated galactose,

where Q is H or a physiologically acceptable salt or CrC₈ alkanyl, C₁-C₈alkenyl, C₁-C₈ alkynyl, aryl, heteroaryl, (CH₂)_(n)-aryl or(CH₂)_(n)-heteroaryl where n is 1-10, and where R⁹ is aryl, heteroaryl,cyclohexane, t-butane, adamantane, or triazole, and any of R⁹ may besubstituted with one to three independently selected of Cl, F, CF₃,C₁-C₈ alkoxy, NO₂, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or OY,C(═O)OY, NY₂ or C(═O)NHY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl.C₁-C₈ alkynyl or C₁-C₁₄ aryl; or

where R¹⁰ is one of

where Q is H or a physiologically acceptable salt, C₁-C₈ alkanyl, C₁-C₈alkenyl, C₁-C₈ alkynyl, aryl, heteroaryl, (CH₂)_(m)-aryl or(CH₂)_(m)-heteroaryl where m is 1-10, n=1-4, Z and Y═C₁-C₈ alkanyl, CrC₈alkenyl, CrC₈ alkynyl, halogenated CrC₈ alkanyl, aryl and heteroarylsubstituted with Me. OMe, halide, OH; and

R⁵═H, D-mannose, L-galactose. D-arabinose, L-fucose, polyols

where X═CF₃, cyclopropyl or phenyl, or

where Q is H or a physiologically acceptable salt,

C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, aryl, heteroaryl,(CH₂)m-aryl or (CH₂)m-heteroaryl where m is 1-10,

and where R¹¹ is aryl, heteroaryl,

where Q is H or a physiologically acceptable salt, Ci-C₈ alkanyl, d-C₈alkenyl, d-C₈ alkynyl, aryl, heteroaryl, (CH₂)_(m)-aryl or(CH₂)_(m)-heteroaryl where m is 1-10, and where n=0-10, and any one ofthe above ring compounds may be substituted with one to threeindependently selected of Cl, F, Ci-C₈ alkanyl, C_(r)C₈ alkenyl, C_(r)C₈alkynyl or OY where Y is H, CrC₈ alkanyl, CrC₈ alkenyl or C_(r)C₈alkynyl.

In some embodiments, the oligosaccharide or glycomimetic compounds arerepresented by the formula:

where Q is H or a physiologically acceptable salt, and Me is methyl.

In other embodiments, the oligosaccharide or glycomimetic compounds arerepresented by the formula:

where Q is H or a physiologically acceptable salt, Me is methyl and Bzis benzoyl.

In still other embodiments, the oligosaccharide or glycomimeticcompounds are represented by the formula:

where Q is H or a physiologically acceptable salt, Me is methyl and Bzis benzoyl.

In other embodiments, the oligosaccharide or glycomimetic compounds arerepresented by the formula:

where Q is H or a physiologically acceptable salt, Me is methyl and Bzis benzoyl.

In still other embodiments, the oligosaccharide or glycomimeticcompounds are represented by the formula:

where Q is H or a physiologically acceptable salt and Me is methyl.

In further embodiments, the oligosaccharide or glycomimetic compoundsare represented by the formula:

where Q is H or a physiologically acceptable salt, Me is methyl and Bzis benzoyl.

In other embodiments, the oligosaccharide or glycomimetic compounds arerepresented by the formula:

where Me is methyl.

In still other embodiments, the oligosaccharide or glycomimeticcompounds are represented by the formula:

where Q is H or a physiologically acceptable salt, Me is methyl and Bzis benzoyl.

In other embodiments, the oligosaccharide or glycomimetic compounds arerepresented by the formula:

where Q is H or a physiologically acceptable salt, Me is methyl and Bzis benzoyl.

Still other embodiments of the oligosaccharide or glycomimetic compoundsare represented by the formula:

where Q is Ht or a physiologically acceptable salt, Me is methyl and Bzis benzoyl.

Yet other embodiments of the oligosaccharide or glycomimetic compoundsare represented by the formula:

where Q is H or a physiologically acceptable salt and Me is methyl andBz is

Other embodiments of the oligosaccharide or glycomimetic compounds arerepresented by the formula:

where Q is H or a physiologically acceptable salt, Me is methyl and Bzis benzoyl.

In other embodiments, the oligosaccharide or glycomimetic compounds arerepresented by the formula:

where Me is methyl, Et is ethyl and Bz is benzoyl.

In still other embodiments, the oligosaccharide or glycomimeticcompounds are represented by the formula:

where Me is methyl and Bz is benzoyl.

Other embodiments of the oligosaccharide or glycomimetic compounds arerepresented by the formula:

where Me is methyl, Et is ethyl and Bz is benzoyl.

Still other embodiments of the oligosaccharide or glycomimetic compoundsare represented by the formula:

where Me is methyl and Bz is benzoyl.

Alternative carbohydrate inhibitors by Magnani et al. are disclosed inInternational Publication No. WO 2007/028050, which is expresslyincorporated herein by reference in its entirety. These compounds arerepresented by the formula:

wherein:

where n=0-2, and R⁸ are independently selected where n=2;

R²═H, —C(═O)OX where X is C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl orC₁-C₁₄ aryl, —C(═O)NH(CH₂)NH₂, —[C(═O)NH(CH₂)_(n)NHC(═O)]_(m)(L)_(m)Z,where n=0-30, m=0-1, L is a linker, and Z is a benzyl amino sulfonicacid, a benzyl amino carboxylic acid, a polyethylene glycol, or a secondcompound or salt thereof having the above formula to form a dimer whereR² of the second compound or salt thereof has m=0, no Z, and is thepoint of attachment,

—O—C(═O)—X, —NH₂, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2 and X isindependently selected from C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,

and any of the above ring compounds may be substituted with one to threeindependently selected of Cl, F, C₁-C₈ alkanyl, C₁-C₈ alkenyl. C₁-C₈alkynyl, C₁-C₁₄ aryl, or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl,C₁-C₈ alkynyl, or C₁-C₁₄ aryl;

6′sulfated GIcNAc, 6′carboxylated GIcNAc, 6′sulfated GaINAc, 6′sulfatedgalactose, 6′carboxylated galactose or

where R⁹ is aryl, heteroaryl, cyclohexane, t-butane, adamantane, ortriazole, and any of R⁹ may be substituted with one to threeindependently selected of Cl, F. C₁-C₈ alkanyl, CrC₈ alkenyl, Ci-C₈alkynyl or OY where Y is H, C₁-C₈ alkanyl, CrC₈ alkenyl, CrC₈ alkynyl orC₁-C₁₄ aryl;

R⁵═H, or R⁴ and R are taken together to form

where R¹⁰ is aryl, heteroaryl,

where n=0-10, and any one of the above ring compounds may be substitutedwith one to three independently selected of Cl, F, C₁-C₈ alkanyl, C₁-C₈alkenyl, C₁-C₈ alkynyl or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenylor C₁-C₈ alkynyl;

R⁶═H, fucose, mannose, arabinose, galactose or polyols;

R⁷═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl or

R⁸═H, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl,

where n=0-3 and X is independently selected from H, OH, Cl, F, N₃, NH₂,C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈ alkynyl, C₁-C₁₄ aryl, OC₁-C₈alkanyl, OC₁-C₈ alkenyl. OC₁-C₈ alkynyl, and OC₁-C₁₄ aryl, and any ofthe above ring compounds may be substituted with one to threeindependently selected of Cl, F, C₁-C₈ alkanyl, C₁-C₈ alkenyl, C₁-C₈alkynyl, C₁-C₁₄ aryl or OY where Y is H, C₁-C₈ alkanyl, C₁-C₈ alkenyl,C₁-C₈ alkynyl, or C₁-C₁₄ aryl.

Alternate carbohydrate inhibitors by Magnani et al. are disclosed in USAppl. Pub. No. 2006/0194745, which is expressly incorporated herein byreference in its entirety. These compounds have the formula:

where n is 0 or 1; X¹ is —PO₂M, —SO₂M or —CF₂— wherein M is apharmaceutically acceptable counterion; R¹ is —OH. —F or —CO₂R⁴ where R⁴is —H or —(CH₂)_(m)—CH₃ and m is 0 to 3; R² is —H, —PO₃M₂, —SO₃M₂,—CH₂—P₃M₂, —CH₂—SO₃M₂, —CF₃, —(CH₂)_(m)—C(R⁶)H—R⁵ or R⁹—N(R¹⁰)— whereinM is defined as above; R³ is —H, —(CH₂)_(m)—C(R⁶)H—R⁵ or R⁹—N(R¹⁰)—where R⁵ and R⁶ are independently selected from —H, —CO₂—R⁷ and —NH—R⁸;R⁷ and R⁸ are independently selected from hydrogen, an alkyl group, anaromatic group, an amino group and a carboxy group, and R⁹ and R¹⁰ areindependently selected from —H, —(CH₂)_(m)—CH₃; —CH₂—Ar, and —CO—Ar,where m is 0 to 3 and Ar is an aromatic group; or

where R₁ and R₂ are independently selected from hydrogen, an alkylgroup, an aromatic group, an amino group or a carboxy group, and —CO—R₃where R₃ is as defined above; and M is a pharmaceutically acceptablecounterion.

Other carbohydrate inhibitors by Magnani et al. are disclosed inInternational Publication No. WO 2006/127906, which is expresslyincorporated herein by reference in its entirety. These compounds havethe formula:

wherein: n=0-20

a benzyl amino sulfonic acid, a benzyl amino carboxylic acid, or asecond compound or salt thereof having the above formula to form adimer:

—O—C(═O)—X or —NH—C(═O)—X

where X is

where n=0-10, and any of the above ring compounds may be substitutedwith one to three of Cl, F, C₁-C₈ alkanyl or OY where Y is H or C₁-C₈alkanyl;

R³═OH,

where R⁴ is cyclohexane, t-butane, adamantane, benzene, triazole, ortriazole substituted with one to three of Cl, F₁ C₁-C₈ alkanyl or OYwhere Y is H or C₁-C₈ alkanyl, and where R⁵ is

where n=0-10, and any one of the above ring compounds may be substitutedwith one to three of Cl, F, C₁-C₈ alkanyl or OY where Y is H or C₁-C₈alkanyl; and

with the proviso that where R¹ is a benzyl amino sulfonic acid and R² orX of R² is aromatic, then R⁴ of R³ is not cyclohexane.

Alternative carbohydrate inhibitors by Magnani et al. are disclosed inInternational Publication No. WO 2005/054264, which is expresslyincorporated herein by reference in its entirety. These compounds arerepresented by the formula:

wherein:

R═H or a benzyl amino sulfonic acid:

R′=a benzyl amino sulfonic acid,

R″=a benzyl amino sulfonic acid, —OH, —OC(═O)—NH—CH₂—CH₃.

wherein the compound possesses a benzyl amino sulfonic acid at R, R′ orR″ but not at more than one of R, R′ and R″.

Still other carbohydrate inhibitors by Magnani et al. are disclosed inInternational Publication No. WO 2005/051920, which is expresslyincorporated herein by reference in its entirety. These compounds arerepresented by the formula:

where L is a linker, which is suitably selected from:

In other illustrative examples, the carbohydrate inhibitor is selectedfrom the selective E-selectin antagonist compounds disclosed by Magnaniet al. in International Publication No. WO 2004/004636, which isexpressly incorporated herein by reference in its entirety. Non-limitingexamples of these compounds are selected from:

Further carbohydrate inhibitors by Magnani et al. are disclosed inInternational Publication No. WO 2003/097658, which is expresslyincorporated herein by reference in its entirety. These compounds arerepresented by a formula selected from the group consisting of:

Still other carbohydrate inhibitors by Magnani et al. are disclosed inU.S. Pat. No. 7,361,644, which is expressly incorporated herein byreference in their entirety. These compounds are selected from thefollowing formulas:

wherein L is a linker.

Representative linkers according to these examples are selected from:

Other carbohydrate inhibitors by Magnani et al. are disclosed in U.S.Pat. No. 7,060,685, which is expressly incorporated herein by referencein their entirety. These compounds consist of a benzyl amino sulfonicacid (BASA) linked to a carbohydrate or a glycomimetic, wherein thecarbohydrate or the glycomimetic binds a selectin; wherein the BASA is

and wherein the carbohydrate or glycomimetic is:

Non-limiting examples of such compounds include:

Still other carbohydrate inhibitors by Magnani et al. are disclosed inU.S. Pat. Nos. 6,121,233, 6,387,884 or 6,391,857, which are expresslyincorporated herein by reference in their entirety. These compounds arerepresented by a formula selected from the group consisting of:

isomers of sialyl Le^(a) or di-sialyl Le^(a);

saccharides that include or consist of carbohydrate portions of sialylLe^(a) or di-sialyl Le^(a); and

glycoconjugates that include a carbohydrate portion of sialyl Le^(a) ordi-sialyl Le^(a),

wherein:

Neu5Ac represents sialic acid; Gal represents galactose; GlcNAcrepresents N-acetyl-glucosamine: Fuc represents fucose and R istypically a ceramide (with a glucose residue interposed) or a protein.

Illustrative examples of isomers of the above compounds include sialylLe^(x), which is an isomer of sialyl Le^(a) wherein the Gal-GlcNAclinkage is β1-4 and the Fuc-GlcNAc linkage is α1→3.

Representative saccharides include the carbohydrate portion of sialylLe^(a) or di-sialyl Le^(a) (i.e., the above structures minus R), andderivatives of either, including those which cross-react with bothsialyl Le^(a) and sialyl Le^(x)

Non-limiting examples of glycoconjugates may be represented by thefollowing structures:

wherein:

R includes H, OH, lipid, ceramide, or one or more amino acids; x, y andz are independently selected from saccharides, or either y or z or bothmay be absent.

In still other embodiments, the carbohydrate inhibitor is selected fromfluorinated glucosamine analogs as disclosed for example by Sackstein elal. in US Pat. Appl. Pub. No. 2006/0281708, which is expresslyincorporated herein by reference in its entirety. Representative analogsof this type are fluorinated N-acetylglucosamines, illustrative examplesof which include2-acetamido-2-deoxy-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranoseand2-acetamido-2-deoxy-1,4,6-tri-O-acetyl-3-deoxy-3-fluoro-D-glucopyranose.

In other illustrative examples, the carbohydrate inhibitor is selectedfrom the selective E-selectin antagonist compounds described by Thoma etal. (2001, Bioorg. Med. Chem. Letts. 11: 923-925), which is expresslyincorporated herein by reference in its entirety. These compounds areselected from the following formulas:

In still other illustrative examples, the carbohydrate inhibitor isselected from the selective E-selectin antagonist compounds described byAli et al. (FASEB J. 2004 January; 18(1):152-4), which is expresslyincorporated herein by reference in its entirety. Non-limiting examplesof these compounds include monovalent E-selectin antagonists selectedfrom:

as well as multivalent-selectin antagonists selected from:

polylysine-a sialyl Lewis^(x) mimetic conjugates as represented by theformula:

wherein x=1-100.

The invention not only encompasses known E-selectin antagonists but alsoantagonists identified by any suitable screening assay. Accordingly, thepresent invention extends to methods of screening for modulatory agentsthat reduce the level or functional activity of E-selectin for use inthe therapeutic or prophylactic methods and compositions of the presentinvention. In some embodiments, the methods comprise: (1) contacting apreparation with a test agent, wherein the preparation contains (i) apolypeptide comprising an amino acid sequence corresponding to at leasta biologically active fragment of an E-selectin polypeptide, or to avariant or derivative thereof; or (ii) a polynucleotide comprising atleast a portion of a genetic sequence that regulates the level orfunctional activity of the E-selectin polypeptide, which is operablylinked to a reporter gene; and (2) detecting a change in the leveland/or functional activity of the E-selectin polypeptide, or anexpression product of the reporter gene, relative to a normal orreference level and/or functional activity in the absence of the testagent, which indicates that the agent modulates the level or functionalactivity of the E-selectin.

Modulators falling within the scope of the present invention includeantagonists of the level or functional activity of E-selectin, includingantagonistic antigen-binding molecules, and inhibitor peptide fragments,antisense molecules, ribozymes, RNAi molecules and co-suppressionmolecules as well as carbohydrate inhibitors of E-selectin function, asfor example described above.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 Dalton.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,desirably at least two of the functional chemical groups. The candidateagent often comprises cyclical carbon or heterocyclic structures oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including, but not limited to: peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analoguesor combinations thereof.

Small (non-peptide) molecule modulators of a E-selectin polypeptide areparticularly advantageous. In this regard, small molecules are desirablebecause such molecules are more readily absorbed after oraladministration, have fewer potential antigenic determinants, or are morelikely to cross the cell membrane than larger, protein-basedpharmaceuticals. Small organic molecules may also have the ability togain entry into an appropriate cell and affect the expression of a gene(eg by interacting with the regulatory region or transcription factorsinvolved in gene expression); or affect the activity of a gene byinhibiting or enhancing the binding of accessory molecules.

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc to produce structural analogues.

Screening may also be directed to known pharmacologically activecompounds and chemical analogues thereof.

Screening for modulatory agents according to the invention can beachieved by any suitable method. For example, the method may includecontacting a cell expressing a polynucleotide corresponding to anE-selectin gene with an agent suspected of having the modulatoryactivity and screening for the modulation of the level or functionalactivity of a protein encoded by the polynucleotide, or the modulationof the level of a transcript encoded by the polynucleotide, or themodulation of the activity or expression of a downstream cellular targetof the protein or of the transcript (hereafter referred to as targetmolecules). Detecting such modulation can be achieved utilizingtechniques including, but not restricted to, ELISA, cell-based ELISA,inhibition ELISA, Western blots, immunoprecipitation, slot or dot blotassays, immunostaining, RIA, scintillation proximity assays, fluorescentimmunoassays using antigen-binding molecule conjugates or antigenconjugates of fluorescent substances such as fluorescein or rhodamine,Ouchterlony double diffusion analysis, immunoassays employing anavidin-biotin or a streptavidin-biotin detection system, and nucleicacid detection assays including reverse transcriptase polymerase chainreaction (RT-PCR).

It will be understood that a polynucleotide from which an E-selectinpolypeptide is regulated or expressed may be naturally occurring in thecell which is the subject of testing or it may have been introduced intothe host cell for the purpose of testing. In addition, thenaturally-occurring or introduced polynucleotide may be constitutivelyexpressed—thereby providing a model useful in screening for agents whichdown-regulate expression of an encoded product of the sequence whereinthe down regulation can be at the nucleic acid or expression productlevel. Further, to the extent that a polynucleotide is introduced into acell, that polynucleotide may comprise the entire coding sequence thatcodes for an E-selectin polypeptide or it may comprise a portion of thatcoding sequence (e.g., the ligand-binding domain of an E-selectinpolypeptide) or a portion that regulates expression of an E-selectingene (e.g., an E-selectin promoter). For example, the promoter that isnaturally associated with the polynucleotide may be introduced into thecell that is the subject of testing. In this instance, where only thepromoter is utilized, detecting modulation of the promoter activity canbe achieved, for example, by operably linking the promoter to a suitablereporter polynucleotide including, but not restricted to, greenfluorescent protein (GFP), luciferase, β-galactosidase and catecholamineacetyl transferase (CAT). Modulation of expression may be determined bymeasuring the activity associated with the reporter polynucleotide.

These methods provide a mechanism for performing high throughputscreening of putative modulatory agents such as proteinaceous ornon-proteinaceous agents comprising synthetic, combinatorial, chemicaland natural libraries. These methods will also facilitate the detectionof agents which bind either the polynucleotide encoding the targetmolecule or which modulate the expression of an upstream molecule, whichsubsequently modulates the expression of the polynucleotide encoding thetarget molecule. Accordingly, these methods provide a mechanism ofdetecting agents that either directly or indirectly modulate theexpression or activity of a target molecule according to the invention.

In some embodiments, the present invention provides assays foridentifying small molecules or other compounds (i.e., modulatory agents)which are capable of inhibiting the level or functional activity ofE-selectin. The assays may be performed in vitro using non-transformedcells, immortalized cell lines, or recombinant cell lines. In addition,the assays may detect the presence of increased or decreased expressionof genes or production of proteins on the basis of increased ordecreased mRNA expression (using, for example, nucleic acid probes thathybridise to an E-selectin gene or coding sequence), increased ordecreased levels of E-selectin (using, for example, antigen bindingmolecules that are immuno-interactive with an E-selectin polypeptide),or increased or decreased levels of expression of a reporter gene (e.g.,GFP, β-galactosidase or luciferase) operably linked to an E-selectinregulatory region (e.g., a promoter or enhancer) in a recombinantconstruct.

Thus, for example, one may culture cells which produce an E-selectinpolypeptide and add to the culture medium one or more test compounds.After allowing a sufficient period of time (e.g., 6-72 hours) for thecompound to inhibit the level or functional activity of the E-selectinpolypeptide, any change in the level from an established baseline may bedetected using, for example, any of the techniques described herein orknown in the art. In specific embodiments, the cells are hemopoieticstem cells. Using suitable nucleic acid probes or antigen-bindingmolecules, detection of changes in the level and or functional activityof an E-selectin expression product, and thus identification of thecompound as agonist or antagonist of the target molecule, requires onlyroutine experimentation.

In some embodiments, recombinant assays are employed in which a reportergene encoding, for example, GFP, β-galactosidase or luciferase isoperably linked to the 5′ regulatory regions of an E-selectin gene. Suchregulatory regions may be easily isolated and cloned by one of ordinaryskill in the art. The reporter gene and regulatory regions are joinedin-frame (or in each of the three possible reading frames) so thattranscription and translation of the reporter gene may proceed under thecontrol of the regulatory elements of the E-selectin gene. Therecombinant construct may then be introduced into any appropriate celltype although mammalian cells are desirable, and human cells are moredesirable. The transformed cells may be grown in culture and, afterestablishing the baseline level of expression of the reporter gene, testcompounds may be added to the medium. The ease of detection of theexpression of the reporter gene provides for a rapid, high throughputassay for the identification of E-selectin antagonists of the invention.

Compounds identified by this method will have potential utility inmodifying the expression of E-selectin in vivo. These compounds may befurther tested in the animal models to identify those compounds havingthe most potent in vivo effects. In addition, as described above withrespect to small molecules having target polypeptide binding activity,these molecules may serve as “lead compounds” for the furtherdevelopment of pharmaceuticals by, for example, subjecting the compoundsto sequential modifications, molecular modeling, and other routineprocedures employed in rational drug design.

In other embodiments, random peptide libraries consisting of a largenumber of possible combinations of amino acids attached to a solid phasesupport may be used to identify peptides that are able to bind to anE-selectin polypeptide or to a functional domain thereof. Identificationof molecules that are able to bind to an E-selectin polypeptide may beaccomplished by screening a peptide library with a recombinant solubleE-selectin polypeptide. The E-selectin polypeptide may be purified,recombinantly expressed or synthesised by any suitable technique. Suchpolypeptides may be conveniently prepared by a person skilled in the artusing standard protocols as for example described in Sambrook, et al.,(1989, supra) in particular Sections 16 and 17; Ausubel et al.,(“Current Protocols in Molecular Biology”, John Wiley & Sons Inc.1994-1998), in particular Chapters 10 and 16; and Coligan et al.,(“Current Protocols in Immunology”, (John Wiley & Sons, Inc, 1995-1997),in particular Chapters 1, 5 and 6. Alternatively, an E-selectinpolypeptide or a portion thereof may be synthesized using solutionsynthesis or solid phase synthesis as described, for example, in Chapter9 of Atherton and Shephard (supra) and in Roberge et al (1995, Science269: 202).

To identify and isolate the peptide/solid phase support that interactsand forms a complex with the E-selectin polypeptide it may be necessaryto label or “tag” the E-selectin polypeptide. In this regard, theE-selectin polypeptide can be conjugated to any suitable reportermolecule, including enzymes such as alkaline phosphatase and horseradishperoxidase and fluorescent reporter molecules such as fluoresceinisothiocyanate (FITC), phycoerythrin (PE) and rhodamine. Conjugation ofany given reporter molecule, with an E-selectin polypeptide, may beperformed using techniques that are routine in the art. Alternatively,E-selectin expression vectors may be engineered to express a chimericE-selectin polypeptide containing an epitope for which a commerciallyavailable antigen-binding molecule exists. The epitope specificantigen-binding molecule may be tagged using methods known in the artincluding labeling with enzymes, fluorescent dyes or colored or magneticbeads.

For example, the “tagged” E-selectin polypeptide conjugate is incubatedwith the random peptide library for 30 minutes to one hour at 22° C. toallow complex formation between E-selectin polypeptide and peptidespecies within the library. The library is then washed to remove anyunbound E-selectin polypeptide. If the E-selectin polypeptide has beenconjugated to alkaline phosphatase or horseradish peroxidase the wholelibrary is poured into a petri dish containing a substrate for eitheralkaline phosphatase or peroxidase, for example,5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3′,4,4″-diamnobenzidine(DAB), respectively. After incubating for several minutes, thepeptide/solid phase-E-selectin polypeptide complex changes color, andcan be easily identified and isolated physically under a dissectingmicroscope with a micromanipulator. If a fluorescently tagged E-selectinpolypeptide has been used, complexes may be isolated by fluorescentactivated sorting. If a chimeric target polypeptide having aheterologous epitope has been used, detection of the peptide/E-selectinpolypeptide complex may be accomplished by using a labeled epitopespecific antigen-binding molecule. Once isolated, the identity of thepeptide attached to the solid phase support may be determined by peptidesequencing.

3.2 Mobilizers of Hematopoietic Stem Cells or Progenitor Cells

Several classes of agents have been shown to increase the circulation ofprogenitor and stem cells by “mobilizing” them from the marrow into theperipheral blood. These include agents that decrease the expression orfunction of a chemokine (the function being the binding of the chemokineto its receptor and further signaling), particularly CXCL12, as well asthose that block or antagonize the chemokine receptor, CXCR4.

Accordingly, in some embodiments, the mobilization agent may be an agentthat decreases the expression or function of a chemokine, moreparticularly, CXCL12, also known as SDF-1. The human amino acid sequenceof SDF-1 comprises the sequence:

MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKRFKM [SEQ ID NO: 223], whichcorresponds to GenBank accession number NP_000600. The alpha isoform hasGenBank accession number NP 954637. The beta isoform has GenBankaccession number NP_000600. The gamma isoform has GenBank accessionnumber NP_001029058.

Alternatively, the mobilization agent may be an agent that blocks orantagonizes a chemokine receptor, in particular, CXCR4. The human aminoacid sequence of CXCR4 comprises the sequence:

MEGISSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS [SEQ IDNO: 224], which corresponds to GenBank accession number CAA12166.

Chemokines are a superfamily of chemoattractant proteins. Chemokinesregulate a variety of biological responses and they promote therecruitment of multiple lineages of leukocytes and lymphocytes to a bodyorgan tissue. Chemokines may be classified into two families accordingto the relative position of the first two cysteine residues in theprotein. In one family, the first two cysteines are separated by oneamino acid residue, the CXC chemokines, and in the other family thefirst two cysteines are adjacent, the CC chemokines. Two minor subgroupscontain only one of the two cysteines (C) or have three amino acidsbetween the cysteines (CX3C). In humans, the genes of the CXC chemokinesare clustered on chromosome 4 (with the exception of SDF-1 gene, whichhas been localized to chromosome 10) and those of the CC chemokines onchromosome 17.

The molecular targets for chemokines are cell surface receptors. Onesuch receptor is CXC chemokine receptor 4 (CXCR4), which is a 7transmembrane protein, coupled to GI and was previously called LESTR(Loetscher, M., Geiser, T., O'Reilly, T., Zwahlen, R., Baggionlini, M.,and Moser, B., (1994) J. Biol. Chem, 269, 232-237), HUMSTR (Federsppiel.B., Duncan, A. M. V., Delaney. A., Schappert. K., Clark-Lewis. I., andJirik, F. R. (1993) Genomics 16, 707-712) and Fusin (Feng, Y., Broeder,C. C., Kennedy, P. E., and Berger, E. A. (1996) HIV-1 entry cofactor:Functional cDNA cloning of a seven-transmembrane G protein-coupledreceptor, Science 272, 872-877). CXCR4 is widely expressed on cells ofhematopoietic origin, and is a major co-receptor with CD4 for humanimmunodeficiency virus 1 (HIV-1) (Feng, Y., Broeder, C. C., Kennedy, P.E., and Berger, E. A. (1996) HIV-1 entry cofactor: Functional cDNAcloning of a seven-transmembrane G protein-coupled receptor, Science272, 872-877).

Chemokines are thought to mediate their effect by binding to seventransmembrane G protein-coupled receptors, and to attract leukocytesubsets to sites of inflammation (Baglionini et al. (1998) Nature 392:565-568). Many of the chemokines have been shown to be constitutivelyexpressed in lymphoid tissues, indicating that they may have ahomeostatic function in regulating lymphocyte trafficking between andwithin lymphoid organs (Kim and Broxmeyer (1999) J. Leuk. Biol. 56:6-15).

Stromal cell derived factor one (SDF-1), also known as CXCL12, is amember of the CXC family of chemokines that has been found to beconstitutively secreted from the bone marrow stroma (Tashiro, (1993)Science 261, 600-602). The human and mouse SDF-1 predicted proteinsequences are approximately 92% identical. Stromal cell derivedfactor-1α (SDF-1α) and stromal cell derived factor-1β (SDF-1 β) areclosely related (together referred to herein as SDF-1). The native aminoacid sequences of SDF-1α and SDF-1β are known, as are the genomicsequences encoding these proteins (see U.S. Pat. No. 5,563,048 issued 8Oct. 1996, and U.S. Pat. No. 5,756,084 issued 26 May 1998).Identification of genomic clones has shown that the alpha and betaisoforms are a consequence of alternative splicing of a single gene. Thealpha form is derived from exons 1-3 while the beta form contains anadditional sequence from exon 4. The entire human gene is approximately10 kb. SDF-1 was initially characterized as a pre-B cell-stimulatingfactor and as a highly efficient chemotactic factor for T cells andmonocytes (Bieul et al. (1996) J. Exp. Med. 184:1101-1110).

Biological effects of SDF-1 may be mediated by the chemokine receptorCXCR4 (also known as fusin or LESTR), which is expressed on mononuclearleukocytes including hematopoietic stem cells. SDF-1 is thought to bethe natural ligand for CXCR4, and CXCR4 is thought to be the naturalreceptor for SDF-1 (Nagasawza et al. (1997) Proc. Natl. Acad. Sci. USA93:726-732). Genetic elimination of SDF-1 is associated with perinatallethality, including abnormalities in cardiac development, B-celllymphopoiesis, and bone marrow myelopoiesis (Nagasawa et al. (1996)Nature 382:635-637). SDF-1 is functionally distinct from otherchemokines in that it is reported to have a fundamental role in thetrafficking, export and homing of bone marrow progenitor cells (Aiuti,A., et al. (1996) J. Exp. Med. 185, 111-120 and Nagasawa, T., et al.(1996) Nature 382, 635-638). SDF-1 is also structurally distinct in thatit has only about 22% amino acid sequence identity with other CXCchemokines.

Agents that decrease the expression of CXCL12 or that block orantagonize CXCR4 may be selected from small organic molecules,polypeptides, nucleic acids and carbohydrates. In more particularembodiments, the polypeptides that decrease the expression of CXCL12 maybe selected from the group consisting of a cytokine, a colonystimulating factor, a protease or a chemokine other than CXCL12. Thecytokine may be selected from the group consisting of interleukin-1(IL-1), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-11(IL-11), interleukin-7 (IL-7) and interleukin-12 (IL12). The proteasemay be selected from the group consisting of a metalloproteinase (likeMMP2 or MMP9) a serine protease, (like cathepsin G, or elastase) acysteine protease (like cathepsin K) and a dipeptidyl peptidase-1 (DDP-1OR CD26). The chemokine other than CXCL12 may be selected from the groupconsisting of IL-8, MIP-1α and Groβ. The colony stimulating factor maybe selected from the group consisting of granulocyte colony stimulatingfactor (G-CSF), granulocyte-macrophage colony stimulating factor(GM-CSF), macrophage colony stimulating factor (M-CSF), stem cellfactor, FLT-3 ligand or a combination thereof. The nucleic acid may be aDNA or an RNA molecule. The nucleic acid may be a small interfering RNA(siRNA) molecule or an antisense molecule specific for CXCL12 or CXCR4.The carbohydrate may be a sulfated carbohydrate selected from the groupconsisting of Fucoidan and sulfated dextran.

4. Therapeutic and Prophylactic Uses

In accordance with the present invention, it is proposed that agentsthat antagonize E-selectin function are useful as actives for enhancingthe hemopoietic properties of mobilizers of hematopoietic stem cells andprogenitor cells. Thus, an E-selectin antagonist can be administered toan individual in combination (e.g., in the same formulation or inseparate formulations) with a mobilizer (“combination therapy”) toenhance hematopoiesis including the mobilization of hematopoietic stemcells and progenitor cells and more particularly to increase the numberof hematopoietic stem cells, progenitor cells and granulocytes such asneutrophils in a patient. The dosages of E-selectin antagonist andmobilizer to be administered may depend on the subject to be treatedinclusive of the age, sex, weight and general health condition thereof.The dosages will also take into consideration the binding affinity ofthe E-selectin antagonist to its target molecule, the hematopoieticcapacity of the mobilizer, their bioavailability and their in vivo andpharmacokinetic properties. In this regard, precise amounts of theagents for administration can also depend on the judgment of thepractitioner. In determining the effective amount of the agents to beadministered in the treatment of an immunocompromised condition, thephysician or veterinarian may evaluate the progression of the disease orcondition over time. In any event, those of skill in the art may readilydetermine suitable dosages of the agents of the invention without undueexperimentation. The dosage of the actives administered to a patientshould be sufficient to effect a beneficial response in the patient overtime such as enhanced hematopoiesis or a reduction in the symptomsassociated with the immunocompromised condition, including a reductionin anemia, thrombocytopenia, agranulocytosis and/or neutropenia. Thedosages may be administered at suitable intervals to boost hematopoiesisor ameliorating the symptoms of the immunocompromised condition. Suchintervals can be ascertained using routine procedures known to personsof skill in the art and can vary depending on the type of active agentemployed and its formulation. For example, the interval may be daily,every other day, weekly, fortnightly, monthly, bimonthly, quarterly,half-yearly or yearly.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active agent which are sufficient to maintainE-selectin-inhibitory effects and hematopoietic function enhancingeffects. Usual patient dosages for systemic administration range from1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150mg/day. Stated in terms of patient body weight, usual dosages range from0.02-25 mg/kg/day, commonly from 0.02-3 mg/kg/day, typically from0.2-1.5 mg/kg/day. Stated in terms of patient body surface areas, usualdosages range from 0.5-1200 mg/m²/day, commonly from 0.5-150 mg/m²/day,typically from 5-100 mg/m²/day.

Thus, the E-selectin antagonist and the mobilizer may be provided ineffective amounts to stimulate or enhance hematopoiesis. This processmay involve administering the E-selectin antagonist separately,simultaneously or sequentially with the mobilizer. In some embodiments,this may be achieved by administering a single composition orpharmacological formulation that includes both agents, or byadministering two separate compositions or formulations at the sametime, wherein one composition includes the E-selectin antagonist and theother, the mobilizer. In other embodiments, the treatment with theE-selectin antagonist may precede or follow the treatment with themobilizer by intervals ranging from minutes to days. In embodimentswhere the E-selectin antagonist is applied separately to the mobilizer,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the E-selectinantagonist would still be able to exert an advantageously combinedeffect on hematopoiesis with the mobilizer, in particular, to maintainor enhance a subject's capacity to mobilize hematopoietic stem cells andprogenitor cells and to increase the number of granulocytes such asneutrophils. In such instances, it is contemplated that one wouldadminister both modalities within about 1-12 hours of each other and,more suitably, within about 2-6 hours of each other. In some situations,it may be desirable to extend the time period for treatmentsignificantly, however, where several hours (2, 3, 4, 5, 6 or 7) toseveral days (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It is conceivable that more than one administration of either theE-selectin antagonist or mobilizer will be desired. Various combinationsmay be employed, where the E-selectin antagonist is “A” and themobilizer is “B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B.

Other combinations are contemplated. Again, both agents are delivered toa PP-subject's immune system in a combined amount effective to enhancehematopoiesis as compared to the administration of the same amount ofmobilizer alone.

As note above, the combination therapy of the present invention findsutility inter alia in the treatment or prophylaxis of immunocompromisedconditions resulting from medical treatment that target hematopoieticstem cells, such as treatments that target rapidly dividing cells orthat disrupt the cell cycle or cell division. The E-selectin antagonistand mobilizer may be used therapeutically after the medical treatment ormay be used prophylactically before the treatment is administered ortogether with the medical treatment Accordingly, the present inventioncontemplates further combination therapies which employ both a medicaltreatment that induces an immunocompromised condition and concurrentadministration of an E-selectin antagonist and a mobilizer ofhematopoietic stem cells or progenitor cells.

It is well known that chemotherapy and radiation therapy target rapidlydividing cells and/or disrupt the cell cycle or cell division. Thesetreatments are offered as part of the treating several forms of cancerand autoimmune disease, aiming either at slowing their progression orreversing the symptoms of disease by means of a curative treatment. Insome embodiments, therefore, the combination therapy or prophylaxis willadditionally employ a chemotherapeutic agent, which is suitable selectedfrom cytostatic agents and cytotoxic agents. Non-limiting examples ofcytostatic agents are selected from: (1) microtubule-stabilizing agentssuch as but not limited to taxanes, paclitaxel, docetaxel, epothilonesand laulimalides; (2) kinase inhibitors, illustrative examples of whichinclude Iressa®, Gleevec, Tarceva™, (Erlotinib HCl), BAY-43-9006,inhibitors of the split kinase domain receptor tyrosine kinase subgroup(e.g., PTK787/ZK 222584 and SU11248); (3) receptor kinase targetedantibodies, which include, but are not limited to, Trastuzumab(Herceptin®). Cetuximab (Erbitux®), Bevacizumab (Avastin™), Rituximab(Ritusan®), Pertuzumab (Omnitarg™); (4) mTOR pathway inhibitors,illustrative examples of which include rapamycin and CCI-778; (5)Apo2L/Trail, anti-angiogenic agents such as but not limited toendostatin, combrestatin, angiostatin, thrombospondin and vascularendothelial growth inhibitor (VEGI); (6) antineoplastic immunotherapyvaccines, representative examples of which include activated T-cells,non-specific immune boosting agents (i.e., interferons, interleukins);(7) antibiotic cytotoxic agents such as but not limited to doxorubicin,bleomycin, dactinomycin, daunorubicin, epirubicin, mitomycin andmitozantrone: (8) alkylating agents, illustrative examples of whichinclude Melphalan, Carmustine, Lomustine. Cyclophosphamide, Ifosfamide,Chlorambucil, Fotemustine, Busulfan, Temozolomide and Thiotepa; (9)hormonal antineoplastic agents, non-limiting examples of which includeNilutamide, Cyproterone acetate, Anastrozole. Exemestane, Tamoxifen.Raloxifene, Bicalutamide, Aminoglutethimide, Leuprorelin acetate,Toremifene citrate, Letrozole, Flutamide, Megestrol acetate andGoserelin acetate; (10) gonadal hormones such as but not limited toCyproterone acetate and Medoxyprogesterone acetate; (11)antimetabolites, illustrative examples of which include Cytarabine,Fluorouracil, Gemcitabine, Topotecan, Hydroxyurea, Thioguanine,Methotrexate, Colaspase, Raltitrexed and Capicitabine; (12) anabolicagents, such as but not limited to, Nandrolone; (13) adrenal steroidhormones, illustrative examples of which include Methylprednisoloneacetate, Dexamethasone, Hydrocortisone, Prednisolone and Prednisone:(14) neoplastic agents such as but not limited to Irinotecan.Carboplatin, Cisplatin, Oxaliplatin, Etoposide and Dacarbazine; and (15)topoisomerase inhibitors, illustrative examples of which includetopotecan and irinotecan.

Illustrative cytotoxic agents can be selected from sertenef, cachectin,ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide (TEMODAR™ from Schering-Plough Corporation,Kenilworth, N.J.), cyclophosphamide, heptaplatin, estramustine,improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride,pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, doxorubicin,irofulven, dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride,diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deansino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN 10755,4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunombicin (seeInternational Publication WO 00/50032), methoxtrexate, gemcitabine, andmixture thereof.

In some embodiments, the concurrent administration of the E-selectinantagonist and the mobilizer is used in combination with radiotherapies,such as but not limited to, conformal external beam radiotherapy (10-100Grey given as fractions over 4-8 weeks), either single shot orfractionated, high dose rate brachytherapy, permanent interstitialbrachytherapy, systemic radio-isotopes (e.g., Strontium 89). Inillustrative examples of this type, the radiotherapy is administered incombination with a radiosensitizing agent. Illustrative examples ofradiosensitizing agents include but are not limited to efaproxiral,etanidazole, fluosol, misonidazole, nimorazole, temoporfin andtirapazamine.

Immunocompromised conditions generally lead to pathogenic infections andthus the present invention also extends to the treatment and/orprophylaxis of infections in individuals suffering from animmunocompromised condition, or to treatment of individuals who arelikely to contract such a condition due to treatment known to beassociated with the occurrence of an immunocompromised condition.Accordingly, an immunocompromised condition arising from a medicaltreatment is likely to expose the individual in question to a higherrisk of infection. It is possible according to the invention toprophylactically treat an infection in an individual having theimmunocompromised condition before or during treatments known togenerate such a condition. By prophylactically treating with concurrentadministration of the E-selectin antagonist and the mobilizer (alsoreferred to herein as an “E-selectin antagonist/mobilizer combination”)before or during a treatment known to generate an immunocompromisedcondition it is possible to prevent a subsequent infection or to reducethe risk of the individual contracting an infection manifesting fromthat condition. In some embodiments, therefore, the present inventionextends to combination therapies, which employ both the E-selectinantagonist/mobilizer combination and an anti-infective agent that iseffective against an infection that develops or that has an increasedrisk of developing from an immunocompromised condition resulting from amedical treatment as broadly described above.

The anti-infective drugs is suitably selected from antimicrobials, whichinclude without limitation compounds that kill or inhibit the growth ofmicroorganisms such as viruses, bacteria, yeast, fungi, protozoa, etc,and thus include antibiotics, amebicides, antifungals, antiprotozoals,antimalarials, antituberculotics and antivirals. Anti-infective drugsalso include within their scope anthelmintics and nematocides.Illustrative antibiotics include quinolones (e.g., amifloxacin,cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine,lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin,lomefloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin,tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin;gemifloxacin; and garenoxacin), tetracyclines, glycylcyclines andoxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline,lymecycline, methacycline, minocycline, oxytetracycline, tetracycline,tigecycline; linezolide, eperozolid), glycopeptides, aminoglycosides(e.g., amikacin, arbekacin, butirosin, dibekacin, fortimicins,gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin,spectinomycin, streptomycin, tobramycin), β-lactams (e.g., imipenem,meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine,cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole,cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole,ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam,cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin,cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam,carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin,azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin,dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin,penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin,cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090,CP-0467, BK-218. FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736,CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycins, macrolides (e.g.,azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin,rosaramicin, roxithromycin, troleandomycin), ketolides (e.g.,telithromycin, cethromycin), coumermycins, lincosamides (e.g.,clindamycin, lincomycin) and chloramphenicol.

Illustrative antivirals include abacavir sulfate, acyclovir sodium,amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate,didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium,ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine,nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin,rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate,stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, andzidovudine.

Non-limiting examples of amebicides or antiprotozoals includeatovaquone, chloroquine hydrochloride, chloroquine phosphate,metronidazole, metronidazole hydrochloride, and pentamidine isethionate.Anthelmintics can be at least one selected from mebendazole, pyrantelpamoate, albendazole, ivermectin and thiabendazole. Illustrativeantifungals can be selected from amphotericin B, amphotericin Bcholesteryl sulfate complex, amphotericin B lipid complex, amphotericinB liposomal, fluconazole, flucytosine, griseofulvin microsize,griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, andterbinafine hydrochloride. Non-limiting examples of antimalarialsinclude chloroquine hydrochloride, chloroquine phosphate, doxycycline,hydroxychloroquine sulfate, mefloquine hydrochloride, primaquinephosphate, pyrimethamine, and pyrimethamine with sulfadoxine.Antituberculotics include but are not restricted to clofazimine,cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide,rifabutin, rifampin, rifapentine, and streptomycin sulfate.

It is also known that medical treatments that target rapidly dividingcells and/or disrupt the cell cycle or cell division (e.g., chemotherapyand radiation therapy) are immunocompromising since cells of the immunesystem including hematopoeitic cells are destroyed or substantiallyreduced in number, thus leading to a state of immunosuppressioncharacterized by neutropenia, agranulocytosis, thrombocytopenia and/oranemia. Accordingly, the present invention finds particular utility inthe treatment or prophylaxis of any one or more of these conditions thatmanifest from a medical treatment as broadly noted above.

Anemia, thrombocytopenia, neutropenia and agranulocytosis are frequentlydefined in terms of laboratory measurements indicating a reducedhematocrit (volume percent), a reduced platelet count (per mm³), areduced neutrophil count (per mm³), a reduced total granulocyte (i.e.,neutrophils, basophils and eosinophils) or white blood cell count (permm³), respectively. Methods of determining these values are well knownin the art, including automated as well as manual methods. The lowerlimits of normal for hematocrits and platelet counts in healthynonpregnant humans is somewhat variable, depending on the age and sex ofthe subject, method of determination, and the norms for the laboratoryperforming the measurements. Generally, however, an adult human subjectis said to have anemia when the hematocrit is less than about 37-40%.Likewise, generally an adult human subject is said to havethrombocytopenia when the platelet count is below about 100,000 per mm³.Anemia is also frequently reported in terms of a reduced hemoglobin(g/dL) or red blood cell count (per mm³). Typical lower limits of normalvalues for these in healthy adult humans are 12-13 g/dL and about4.1×10⁶ per mm³, respectively. Generally an adult human subject is saidto have neutropenia when the neutrophil count falls below 1000 per mm³.Additionally, an adult human is generally said to have agranulocytosiswhen the total granulocyte cell count falls below 500 cells/mm³.Corresponding values for all these parameters are different for otherspecies.

Hematopoeitic disorders such as anemia, thrombocytopenia, neutropeniaand agranulocytosis are also frequently associated with clinical signsand symptoms in relation to their degree of severity. Anemia may bemanifested as pallor, generalized fatigue or weakness, reduced exercisetolerance, shortness of breath with exertion, rapid heart rate,irregular heart rhythm, chest pain (angina), congestive heart failure,and headache. Thrombocytopenia is typically manifested in terms ofspontaneous or uncontrolled bleeding, petechiae, and easy bruising.Neutropenia is associated with infections, including notably infectionsfrom endogenous microbial flora, and lack of inflammation.

Accordingly, the present invention contemplates ancillary combinationtherapies which employ both the E-selectin antagonist/mobilizercombination and an ancillary treatment that treats a hematopoeiticdisorder as broadly described above. In some embodiments, the ancillarycombination therapy will employ an E-selectin antagonist/mobilizercombination and a medicament selected from an anemia medicament, athrombocytopenia medicament, an agranulocytosis medicament or aneutropenia medicament, illustrative examples of which include steroids,inducers of steroids, and immunomodulators.

The steroids include, but are not limited to, systemically administeredcorticosteroids including methylprednisolone, prednisolone andprednisone, cortisone, and hydrocortisone. Inducers of steroids include,but are not limited to adrenocorticotropic hormone (ACTH).

Corticosteroids inhibit cytokine production, adhesion proteinactivation, and inflammatory cell migration and activation. The sideeffects associated with systemic corticosteroids include, for instance,reversible abnormalities in glucose metabolism, increased appetite,fluid retention, weight gain, mood alteration, hypertension, pepticulcer, and asceptic necrosis of bone. Some side effects associated withlonger term use include adrenal axis suppression, growth suppression,dermal thinning, hypertension, diabetes mellitus. Cushing's syndrome,cataracts, muscle weakness, and in rare instances, impaired immunefunction. It is recommended that these types of compounds be used attheir lowest effective dose.

Commonly used anemia drugs which are currently on the market or indevelopment include recombinant human EPO (EPOGEN; PROCRIT),preparations of iron (ferrous and ferric, CHROMAGEN; FEOSOL; INFED;IROSPAN; NEPHRO-FER; NEPHRO-VITE; NIFEREX; NU-IRON; SLOW FE), vitaminB12, vitamin B6, folic acid (CHROMAGEN; FERRO-FOLIC; NEPHRO-FER;NIFEREX), ascorbic acid, certain metabolites of vitamin D (calcitrioland alphacalcidol; CALCIJEX; ROCALTROL), androgens, and anabolicsteroids (ANADROL), carnitine. In a specific embodiment the anemiamedicament is recombinant EPO.

Drugs in common usage or development for the treatment ofthrombocytopenia include glucocorticoids (prednisolone; prednisone;methylprednisolone; SOLUMEDROL), recombinant TPO, recombinant MGDF,pegylated recombinant MGDF, and lisophylline. In a specific embodimentthe thrombocytopenia medicament is recombinant TPO.

Drugs in common usage or development for the treatment of neutropeniainclude glucocorticoids (prednisolone; prednisone; methylprednisolone;SOLUMEDROL), immunoglobulin G (SANDOGLOBULIN, IVEEGAM, GAMMAR-P, GAMIMNEN, GAMMAGARD S/D), androgens, recombinant IFN-γ (ACTIMMUNE), anduteroferrin. Antibiotics are frequently administered in association withneutropenia medicaments to treat or reduce the risk of infection.

As noted above, the present invention encompasses co-administration ofan E-selectin antagonist/mobilizer combination in concert with anadditional agent. It will be understood that, in embodiments comprisingadministration of the E-selectin antagonist/mobilizer combination withother agents, the dosages of the actives in the combination may on theirown comprise an effective amount and the additional agent(s) may furtheraugment the therapeutic or prophylactic benefit to the patient.Alternatively, the E-selectin antagonist/mobilizer combination and theadditional agent(s) may together comprise an effective amount forpreventing or treating the immunocompromised condition or infection. Itwill also be understood that effective amounts may be defined in thecontext of particular treatment regimens, including, e.g., timing andnumber of administrations, modes of administrations, formulations, etc.

In other aspects, the present invention also contemplates administeringa high dose of the medical treatment that induces the immunocompromisedcondition, without inducing side effects. Ordinarily, when medicaltreatments such as chemotherapy and radiotherapy are administered in ahigh dose, a variety of side effects can occur, including the inductionof the immunocompromised condition and infection. As a result of theseside effects, the medical treatment is not administered in such highdoses. In accordance with the present invention, such high doses ofmedical treatment (e.g., a higher dose of chemotherapeutic agent orradiation) which ordinarily induce side effects can be administeredwithout inducing the side effects as long as the subject also receivesconcurrent administration of an E-selectin antagonist and a mobilizer ofhematopoietic stem cells or progenitor cells. The type and extent of theside effects ordinarily induced by the medical treatment will depend onthe particular treatment used.

Suitably, the E-selectin antagonist/mobilizer combination, andoptionally the ancillary treatment, are administered on a routineschedule. Alternatively, the ancillary treatment may be administered assymptoms arise. A “routine schedule” as used herein, refers to apredetermined designated period of time. The routine schedule mayencompass periods of time which are identical or which differ in length,as long as the schedule is predetermined. For instance, the routineschedule may involve administration of the im E-selectin antagonist on adaily basis, every two days, every three days, every four days, everyfive days, every six days, a weekly basis, a monthly basis or any setnumber of days or weeks there-between, every two months, three months,four months, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, twelve months, etc. Alternatively,the predetermined routine schedule may involve concurrent administrationof the E-selectin antagonist and the mobilizer on a daily basis for thefirst week, followed by a monthly basis for several months, and thenevery three months after that. Any particular combination would becovered by the routine schedule as long as it is determined ahead oftime that the appropriate schedule involves administration on a certainday.

Additionally, the present invention provides pharmaceutical compositionsfor treating or preventing an immunocompromised condition that resultsfrom a medical treatment as broadly described above. The pharmaceuticalcompositions include an E-selectin antagonist and a mobilizer ofhematopoietic stem cell or progenitor cells, optionally formulated in apharmaceutically acceptable carrier. The pharmaceutical composition mayinclude an ancillary or additional medicament as broadly describedabove. In some embodiments, the E-selectin antagonist and the mobilizerwill be present in the pharmaceutical composition in an effective amountfor preventing or treating an immunocompromised condition (e.g., anemia,thrombocytopenia, or neutropenia). The effective amount for preventingor treating the immunocompromised condition is that amount whichcompletely or partially prevents the development of, prevents theworsening of, or treats the established existence of, theimmunocompromised condition. In some instances, the effective amount forpreventing or treating immunocompromised condition completely orpartially prevents or treats clinical symptoms of that condition.

In addition to clinical outcomes measured in terms of physiology, invitro assays measuring erythrocyte, platelet, granulocyte and totalwhite blood cell counts may be used in determining a therapeuticallyeffective amount of a particular E-selectin antagonist. These methodsare standard medical laboratory techniques that are well known in theart. In common practice such measurements may be made by automated cellcounting devices designed for that purpose, or they may be performedmanually. Manual counts may be more accurate than automated counts whencell counts are particularly low.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients. Depending on the specific conditions being treated, theformulations may be administered systemically or locally. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition. Suitable routes may, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. For injection,the active agents or drugs of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

The drugs can be formulated readily using pharmaceutically acceptablecarriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds of the invention tobe formulated in dosage forms such as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated. These carriers may be selected from sugars,starches, cellulose and its derivatives, malt, gelatin, talc, calciumsulfate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffered solutions, emulsifiers, isotonic saline, andpyrogen-free water.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilisers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatine, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Such compositions may beprepared by any of the methods of pharmacy but all methods include thestep of bringing into association one or more drugs as described abovewith the carrier which constitutes one or more necessary ingredients. Ingeneral, the pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical which can be used orally include push-fit capsules madeof gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

Dosage forms of the drugs of the invention may also include injecting orimplanting controlled releasing devices designed specifically for thispurpose or other forms of implants modified to act additionally in thisfashion. Controlled release of an agent of the invention may be effectedby coating the same, for example, with hydrophobic polymers includingacrylic resins, waxes, higher aliphatic alcohols, polylactic andpolyglycolic acids and certain cellulose derivatives such ashydroxypropylmethyl cellulose. In addition, controlled release may beeffected by using other polymer matrices, liposomes or microspheres.

The drugs of the invention may be provided as salts withpharmaceutically compatible counterions. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thatare the corresponding free base forms.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC50 asdetermined in cell culture (e.g., the concentration of an active agent,which achieves a half-maximal inhibition in activity of an E-selectinpolypeptide). Such information can be used to more accurately determineuseful doses in humans.

Toxicity and therapeutic efficacy of such drugs can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. The dataobtained from these cell culture assays and animal studies can be usedin formulating a range of dosage for use in human. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See for example Fingl et al., 1975, in“The Pharmacological Basis of Therapeutics”, Ch. 1 p 1).

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a tissue, which is preferably subcutaneous or omental tissue, oftenin a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tissue-specific antibody.The liposomes will be targeted to and taken up selectively by thetissue.

In cases of local administration or selective uptake, the effectivelocal concentration of the agent may not be related to plasmaconcentration.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLES Example 1 Hematopoietic Stem Cell Turnover is Delayed 2.7-FoldIn Vivo in the Bone Marrow of Mice Lacking the E-Selectin Gene

Using C57BL/6 mice knocked-out for either or both the P- or E-selectingenes, it was shown that deletion of E-selectin, but not P-selectin,delays hematopoietic stem cell turn-over in the bone marrow in vivo.Mice were fed with BrdU in their drinking water for up to 14 days andsacrificed on days 3, 5, 7 and 14 to sort LSK34− hematopoietic stemcells. Following antibody staining with an anti-BrdU monoclonalantibody, 50% of LKS34-cells from the bone marrow of wild-type (WT) andP-sel−/− mice incorporated BrdU in 3.6 days whereas 50% of LKS34− cellsfrom E-sel−/− and P/E-selectin double KO mice incorporated BrdU in 9.5days (see FIG. 1). Thus, the cycling time of hematopoietic stem cell is2.7 fold slower in the absence of E-selectin.

To determine whether this effect was mediated by the two previouslyidentified E-selectin receptors PSGL-1 and CD44, BrdU incorporationexperiments were repeated with mice knocked-out for both the PSGL-1 andCD44 genes. LKS34− cell turnover in these mice was identical to that ofwild-type suggesting that the effect is mediated by distinct unknownreceptor(s). FIGS. 5 and 6 Confirm that BM HSPC can adhere to E-selectinindependent of PSGL-1 and CD44.

Example 2 In the Absence of E-Selectin, Bone Marrow Hematopoietic StemCells are Metabolically Less Active

To support findings on hematopoietic stem cell turnover with BrdU,lineage-negative Sca1-positive CD117-positive (LSK) hematopoietic stemcells were isolated from the bone marrow and stained with Rhodamine123,a viable dye that binds to mitochondrial membranes and is retained bymetabolically active respirating cells.

A higher proportion of LKS cells from E-selectin−/− mice were rhodaminedull (43±3%) compared to LKS cells from wild-type mice (30±2%; p=0.002)confirming that a greater proportion of hematopoietic stem cells fromE-selectin knock-out mice are quiescent (FIG. 2).

To confirm that metabolically active Rhodamine123 dull cells cycle andincorporate BrdU less rapidly, Rhodamine bright and Rhodamine dull LKScells were sorted from the same bone marrows as shown in FIG. 2.Rhodamine dull LKS cells incorporated BrdU 7 times less than Rhodaminebright LSK cells after 2 days of BrdU feeding to the mice, showing thatless metabolically active Rhodamine dull stem cells are 7 times lesslikely to have cycled (and be BrdU-positive) during the two day periodof BrdU feeding.

-   -   Rho bright LKS+→35% BrdU+ (2 days)    -   Rho dull LKS+→5% BrdU+ (2 days)

Taken together, these results confirm that in the absence of theE-selectin gene, hematopoietic stem cells residing in the bone marroware less metabolically active and divide slower than in wild-type mice.

Example 3 Hematopoietic Stem Cell Turnover is Lower in E-Selectin KOMice Following Cytotoxic Insult with 5-Fluorouracil

To determine the effect of E-selectin gene deletion on hematopoieticstem cell recovery following cytotoxic stress, E-selectin KO andwild-type mice were injected with a single dose of 5-fluorouracil (5FU150 mg/kg). As CD117 is strongly down-regulated in the bone marrow of5FU-treated mice, the proportion of lineage-negative Sca1-positiveCD41-negative CD48-negative CD150-positive long-term reconstitutinghematopoietic stem cells⁶ that incorporate BrdU was measured. For thispropose, mice were sacrificed prior to and at day 3 or day 7 following5-FU injection and BrdU was given continuously through drinking water inthe last 17 hours before sacrificed. BrdU incorporation remainedsignificantly lower in hematopoietic stem cells from E-selectinknock-out mice on days 3 and 7 post-5FU suggesting that the observeddecreased HSC turn-over in the absence of E-selectin may protects themfrom the cytotoxic effect of 5FU (FIG. 3).

The recovery of long-term reconstituting hematopoietic stem cells wasalso enhanced in E-selectin^(−/−) mice at day 7 post-5FU with a 5-foldincrease in HSC numbers per femur compared to WT mice (FIG. 4).

The fact that the proliferation of hematopoietic stem cells was lowerwith increased absolute number of stem cells per femur at day 7following chemotherapy with 5-FU shows that hematopoietic stem cellswere more resistant to the cytotoxic effect of 5FU in E-selectinknock-out mice. Thus deletion of the E-selectin results in enhancedresistance of hematopoietic stem cells to the cytotoxic effect ofchemotherapy.

Example 4 The Effect of E-Selectin on the Turn-Over of HematopoieticStem Cells is not Mediated by the Two Previously Described E-SelectinLigands P-Selectin Glycoprotein Ligand-1 (PSGL-1 or CD162) and CD44

BrdU incorporation data from FIG. 1 show that LSK34− hematopoietic stemcells isolated from the bone marrow of mice lacking both the PSGL-1 andCD44 genes incorporate BrdU and cycle at the same rate as hematopoieticstem cells isolated from wild-type mice. Thus, this suggests that thedelayed cell cycling observed in hematopoietic cells from E-selectin KOmice is not mediated by the two previously described E-selectinreceptors PSGL-1 and CD44¹⁰.

To confirm this further, the inventors measured interaction ofhematopoietic stem cells isolated from mice lacking both PSGL-1 and CD44with recombinant E-selectin. In adhesion assays in 96-well polystyreneplates coated with recombinant proteins made of the entire extracellulardomain of mouse E-selectin, P-selectin or VCAM-1/CD106 fused to the Fcfragment of human IgG1 (muEsel-IgG1Fc, muPsel-IgG1Fc, ormuVCAM1-IgG1Fc), deletion of the PSGL1 gene completely abrogatedadhesion of bone marrow linage-negative CD117-positive hematopoieticprogenitor cells to P-selectin, demonstrating that PSGL1 is the soleP-selectin receptor on bone marrow hematopoietic progenitor cells. Insharp contrast, the deletion of either or both the PSGL1 and CD44 genes,did not alter adhesion of bone marrow hematopoietic progenitor cells toE-selectin or to VCAM-1, an unrelated cell adhesion molecule whosecellular receptors are α4 integrins/CD49d (FIG. 5).

In a second assay, binding of recombinant E-selectin and P-selectin wasdirectly measured in solution by flow cytometry. For this purpose,recombinant human P-selectin or E-selectin extracellular domains fusedwith the Fc portion of human IgM were used as selectins required priorclustering to bind to their cellular receptors. As IgM are decamericproteins, each fusion recombinant protein is a decamer or eitherP-selectin or E-selectin. The clustering resulting from decamerizationof selectin enables them to directly interact in solution with theircellular receptors. The inventors therefore measured the binding ofrecombinant selectin-IgM Fc fusion proteins to bone marrow cellsisolated from wild-type, PSGL1−/−, CD44−/− or PSGL1−/−CD44−/− double KOmice. Detection by flow cytometry was performed by pre-complexing theselectin-IgMFc fusion proteins with a Cy5-conjubated donkey anti-humanIgM antibody. FIG. 6 shows that while deletion of both PSGL1 and CD44genes markedly reduced binding of recombinant E-selectin-IgMFc to bonemarrow granulocytes, the deletion of these two genes did not decreasethe binding of recombinant E-selectin-IgMFc to bone marrow LSKhematopoietic stem cells. Thus these experiments confirm that PSGL1 andCD44 are necessary and sufficient for E-selectin binding to maturegranulocytes, hematopoietic stem cells can bind and adhere to E-selectinvia alternative receptors which are not encoded by the PSGL1 and C44genes.

Example 5 Enhanced Mobilization of Hematopoietic Stem and ProgenitorsCells in Response to G-CSF in Mice Lacking the E-Selectin Gene

Mobilization is the forced movement of HSC from the bone marrow into theblood where they can be easily collected. Mobilized blood is now thepreferred source of HSC for transplantation having surpassed the moretraditional bone marrow aspirates.

Currently, the main use of mobilized HSC is to transplant into patientswho have undergone repeated rounds of chemotherapy to treat cancers (aside-effect of chemotherapy is bone marrow/HSC damage), or to treat somegenetic diseases of the hematopoietic and immune system in order toreconstitute normal disease-free hematopoiesis in these patients.

The main limitation to the success of HSC transplantation however, isobtaining sufficient numbers of HSC from a donor to ensure rapidengraftment and hematopoietic/immune reconstitution. This isparticularly true in the case of autologous transplantation frompatients who have undergone many rounds of chemotherapy. Up to 50% ofpatients who have undergone repeated rounds of high dosechemotherapy/radio therapy are unable to mobilize sufficient HSC intothe blood for a successful transplant (in human patients, usually >2×10⁶CD34⁺ phenotypic cells/kg body weight). In addition, up to 5% of normalhealthy allogeneic donors fail to mobilise sufficient numbers of HSC inresponse to G-CSF in order to ensure rapid and safe haematopoieticreconstitution of transplant recipient.

In accordance with the present invention, the results presented in FIGS.7 and 8 show that mobilization of HSC can be enhanced significantlyfollowing deletion of the cell adhesion molecule E-selectin (CD62E).

In particular, FIG. 7 shows that in steady-state conditions, salineinjected wild-type and E-selectin KO mice have equivalent numbers ofblood leukocytes, no detectable number of CFC and very low levels ofphenotypic Lin-Sca-1+CD117+CD48-CD50⁺ HSC in the peripheral blood. Aftera 3-day course of pegylated G-CSF administration, mobilization is higherin E-selectin KO than in wild-type mice with a 2.5-fold high number ofCFC (FIG. 7B), 2.5-fold high number of phenotypic HSC (FIG. 7 C) and a12-fold higher number of functional long-term reconstituting HSC per mLof mobilized blood.

In order to mimic the clinical setting of autologous transplantationwhere cancer patients are heavily treated with chemotherapy prior tomobilization of their own HSC and transplantation, two cohorts ofwild-type and E-selectin KO mice were injected fortnightly with a singledose of cyclophosphamide for 8 rounds. After the 8^(th) round, micerested 6 weeks prior to mobilization following injection of pegylatedG-CSF. FIG. 8 shows that E-selectin KO mice pre-treated withchemotherapy mobilized 5-fold more CFC and 2.5-fold more phenotypic HSCper mL of peripheral blood, as compared to wild-type mice pre-treatedwith chemotherapy.

Materials and Methods Incorporation of 6-Bromodeoxyurine (BrdU) toMeasure Hematopoietic Stem Cell Turnover In Vivo

The aim of this method is to measure the proportion of hematopoieticstem cells that incorporate the nucleotide analog BrdU into theirgenomic DNA during a given period of time in vivo. Since BrdU can onlyintegrate into genomic DNA during the S phase of the cell cycle, theproportion of cells that have incorporated BrdU is equal to theproportion of cells that have divided or are dividing during the periodof animal feeding with BrdU s.

Adult mice (10-14 week-old) with homologous deletion of the E-selectingene (Sele), the P-selectin gene (Selp), both the E-selectin andP-selectin genes, or both the CD162/PSGL1 (Selplg) and CD44 (Cd44) geneswere given 1 mg/mL BrdU solubilised in their drinking water for acontinuous period of up to 14 days.

At various time-points, mice were sacrificed and femurs, tibias andiliac crest harvested. One cleaned, bones were crushed with a mortar andpestle in phosphate-buffered saline containing 2% new-born calf serum toextract bone marrow cells. Mononucleated bone marrow cells were isolatedby centrifugation at 500×g on a density gradient made with 62.5%Percoll.

Cells expressing c-KIT/CD117 were next enriched by magnetic cell sorting(MACS) using mouse CD117 magnetic microbeads (Miltenyi Biotec). For thispurpose, mononucleated cells at the Percoll interface were washed inphosphate-buffered saline containing 0.5% bovine serum albumin and 2 mMethylene diamine tetraacetate (MACS washing buffer), resuspended at 10⁸cells/mL and subsequently incubated with 1.5 μL mouse CD117 microbeadsper 10⁷ cells. Cells were incubated for 15 minutes on ice with magneticbeads, washed once in MACS washing buffer, pelleted at 440×g,resuspended at 10⁸ cells/mL in MACS washing buffer. Cells expressingCD117 were then enriched by separation on an autoMACS Separator(Miltenyi Biotec) using the cell depletion program.

Hematopoietic stem cells were further isolated from this CD117MACS-enriched population by fluorescence-activated cell sorting (FACS)using the following panel of antibodies: a) biotinylated antibodiesagainst lineage-specific antigens CD3, CD5, B220/CD45R, CD11b,Gr-1/Ly-6G, Ter119 together with streptavidin conjugated to PercP-Cy5.5;b) fluorescein isothiocyanate (FITC) conjugated anti-CD34; c)phycoerythrin (PE) conjugated anti-Sca-1/Ly-6A/E; d) allophycocyanin(APC) conjugated anti-CD117 antibodies. Hematopoietic stem cells withthe phenotype Lineage-negative, Sea-1-positive, CD117-positive andCD34-negative (LSK34−), were sorted on a FACS Aria cell sorter (BDBiosciences), collected in phosphate-buffered saline containing 2%newborn calf serum and cytospun on positively charged glass slides. Oncecytospun on glass slides, LSK34− cells were air dried and fixed with thefixative provided in the BD Pharmingen BrdU detection kit (catalog#551321). Staining with a monoclonal antibody specific for BrdU was thenperformed exactly following the kit instructions. Followingcounterstaining with dilute hematoxylin and mounting with Aquamount, theproportion of cells staining for BrdU was manually counted using amicroscope.

In additional experiments, BrdU incorporation was measured in a purerpopulation of hematopoietic stem cells. This population has thephenotype Lineage-negative, Seal-positive, CD117-positive,CD41-negative, CD48-negative, CD150-positive. This very rare population(0.05% of the bone marrow) has been described to be homogenous with highreconstituting activity. Specifically, 50% of lethally irradiated micetransplanted with a single cell exhibiting this phenotype canreconstitute a full hematopoietic/immune system from this single cell⁶.The combination of antibodies to sort these cells is as follows: a)biotinylated antibodies against lineage-specific antigens CD3, CD5,B220/CD45R, CD11b, Gr-1/Ly-6G, CD41, Ter119 together with streptavidinconjugated to PercP-Cy5.5; b) fluorescein isothiocyanate (FITC)conjugated anti-CD48; c) phycoerythrin (PE) conjugated anti-CD150; d)PECy7 conjugated anti-Sca-1/Ly-6A/E; f) allophycocyanin (APC) conjugatedanti-CD117 antibodies

Determination of Hematopoietic Stem Cell Metabolic Activity by FlowCytometry Using Rhodamine123 Fluorescent Dye

Rhodamine123 is a vital fluorescent dye that incorporates preferentiallyin mitochondria. It has been previously described that most quiescenthematopoietic stem cells with highest reconstituting potential followingtransplant incorporate low levels of rhodamine 123 whereas metabolicallyactive stem cells incorporate high levels of rhodamine 123^(5,7).

For this purpose, bone marrow cells extracted as above were resuspendedat 10⁶/mL in PBS with 2% fetal calf serum and incubated withRhodamine123 (0.1 μg/mL) at 37° C. for 20 min then washed in PBS with 2%fetal calf serum and incubated at 10⁶/mL in PBS+2% serum for another 15min at 37° C. to efflux excess dye incorporated in cells. FollowingRho123 efflux, cells were kept on ice and subsequently stained withbiotinylated lineage antibodies (CD3, CD5, B220, Gr-1, F4/80, Ter119),CD117-APC, Sca-1-PE then washed and incubated withstrepatvidin-PerCPCy5.

Lineage-negative, CD117-positive, Sca1-positive cells were analysed byflow cytometry for Rhodamine123 fluorescence on a BD Biosciences FACSCalibur flow cytometer

Measurement of HSC Cycling and Number In Vivo Following Cytotoxic Insultwith the Chemotherapeutic Drug 5-Fluorouracil (5-Fu)

Adult mice (10-14 week-old) with homologous deletion of the E-selectingene were administered intravenously a single dose of 5-FU at 150 mg/kg.At days 2 and 6 following 5-FU, mice were injected intraperitoneallyBrdU 100 mg/kg followed by a continuous dose of 1 mg/mL in theirdrinking water at a concentration of 1 mg/mL. 18 hours following theinjection of BrdU. Mice were sacrificed, their bone marrow cellscollected as described above and stained with the following combinationof antibodies: a) biotinylated antibodies against lineage-specificantigens CD3, CD5, B220/CD45R. CD11b, Gr-1/Ly-6C/G, CD41, Ter119together with streptavidin conjugated to PercP-Cy5.5; b) fluoresceinisothiocyanate (FITC) conjugated anti-CD48; c) phycoerythrin (PE)conjugated anti-CD150; d) PECy7 conjugated anti-Sca-1/Ly-6A/E; f)allophycocyanin (APC) conjugated anti-CD117 antibodies. Truehematopoietic stem cells with the phenotype Lineage-negative,Sca1-positive, CD117-positive, CD41-negative, CD48-negative,CD150-positive were counted and sorted as described above. Sorted cellswere cytospun on glass slides and stained with anti-BrdU antibodies asdescribed above.

Cell Adhesion Assay of Hematopoietic Progenitor Cells on ImmobilisedRecombinant Mouse E-Selectin and P-Selectin Fusion Proteins

96-well polystyrene cell culture plates were coated overnight at 4*Cwith 50 μL per well of phosphate buffered saline containing 3 μg/ml ofrecombinant proteins made of the entire extracellular domain of mouseE-selectin, P-selectin or VCAM-1/CD106 fused to the Fc fragment of humanIgG1 (muEsel-IgG1Fc, muPsel-IgG1Fc, or muVCAM1-IgG1Fc respectively, fromR&D Systems)) as previously described 8. Prior to the experiment, plateswere flicked to remove excess coating solution and filled with Hepesbuffered saline supplemented with 2% bovine serum albumin to blocknon-specific adhesion to plastic surfaces. Following 1 hour incubationat 37° C., coated wells were washes twice with cell adhesion buffer(Iscove's modified Dulbecco medium supplemented with 0.2% bovine serumalbumin and 1 mM CaCl2).

Bone marrow cells from CD44 KO, PSGL1 KO and CD44-PSGL1 double KO micewere stained with FITC-conjugated biotinylated rat monoclonal antibodiesspecific for CD3, CD5. B220/CD45R, CD11b, Gr1 and Teri 19lineage-specific antigens and PE-conjugated anti-CD117 antibody.Lineage-negative CD117-positive hematopoietic progenitor cells were thensorted by fluorescence activated cell sorting on an Aria cell sorter (BDBiosciences).

Sorted Lineage-negative CD117-positive hematopoietic progenitor cellswhen then washed and resuspended in cell adhesion buffer and labelledwith the intracellular fluorescent dye calcein-AM (Molecular Probes) for40 minutes at 37° C. 8. Following labelling with calcein-AM, cells werewashed in cell adhesion buffer and resuspended at 105 cells/mL. 100 μL(104 cells) were deposited in each well coated with muEsel-IgG1Fc,muPsel-IgG1Fc, muVCAM1-IgG1Fc, or serum albumin alone, centrifuged at200×g for 5 minutes to sediments cells at the bottom of coated wells andfurther incubated for 40 minutes on ice. Following this incubations,non-adherent cells were removed by 4 gentle washes with cell adhesionbuffer. The fluorescence contained in the remaining adherent cells wasmeasured on a Fluorostar plate fluorometer following exciting at 488 nmusing a 530 nm filter.

Measurement of the Binding of Recombinant E-Selectin and P-Selectin onHaematopoietic Stem Cells in Suspension

Recombinant human E-selectin and P-selectin extracellular domains fusedwith the Fc fragment of human IgM (selectin-IgMFc) were produced assupernatants following transfection of COS7 cell line with pCDM8plasmids containing the corresponding cDNA⁹. Following transfection,COS7 medium was replaced by serum-free X-VIVO10 medium and conditionedfor three days post transfection. Saturating doses ofselectin-containing supernatants were determined by flow cytometry usingthe human myeloid cell line KG1a.

Prior to the experiment, recombinant selectin-IgMFc fusion proteins werecomplexed with Cy5-conjugated donkey IgG F(ab)′2 fragments anti-humanIgM. For this purpose, serum-free supernatants were incubated with anequal volume of cell adhesion buffer (Iscove's modified Dulbecco mediumsupplemented with 0.2% bovine serum albumin and 1 mM CaCl₂) containing a1/50 dilution of Cy5-conjugated donkey IgG F(ab)′2 fragments anti-humanIgM (Jackson ImmunoResearch) for 2 hours at 4° C.

Bone marrow cells from CD44 KO, PSGL1 KO and CD44-PSGL1 double KO micewere depleted of lineage-positive cells on an autoMACS Separator usingbiotinylated rat monoclonal antibodies specific for CD3, CD5,B220/CD45R, CD11b, Gr1 and Ter119 lineage-specific antigens andstreptavidin-coated magnetic immunobeads (Miltenyi Biotec). Followingdepletion, lineage-negative bone marrow cells were stained on ice for 40minutes with FITC-conjugated anti-Sca1/Ly6A-E and PE-conjugatedanti-CD117 rat monoclonal antibodies. Following washing with celladhesion buffer described above, 10⁶ labelled lineage-depleted bonemarrow cells were resuspended in a volume of 25 μL of cell adhesionbuffer. Twenty-five μL of selectin-IgMFc pre-complexed withCy5-conjugated donkey anti-human IgM was then added to the cells andfurther incubated for 40 minutes at 4° C. Negative controls wereperformed by adding the calcium chelator ethylene diamine tetraaceticacid (5 mM) in the cell adhesion buffer as selectin-mediatedinteractions are strictly calcium-dependant. Binding of selectin-IgMFcfusion proteins was measured by flow cytometry on a FACS Calibur flowcytometer (BD Biosciences).

Mobilization of HSC with Recombinant Human G-CSF

Mobilization was induced in wild-type C57BL/6 mice and in C57BL/6 micecarrying homologous deletion of the E-selectin gene (E-selectin KOmice), using recombinant human G-CSF following a mobilization dosing andprotocol similar to that used for human patients.

Adult mice (10- to 14-week old males) were administered recombinanthuman pegylated G-CSF (granulocyte colony stimulating factor)subcutaneously. Two doses were given 36 hours apart, each at 0.5 mg/kg.The G-CSF used was Neulasta, from Amgen. Neulasta was diluted to 0.1mg/mL in sterile saline for injection and 100 μL used per injection into20 g mice. In some experiments, a cohort of ‘control’ mice receivedsaline injections alone.

Blood containing the mobilized HSC, was collected 3 days following theinitial G-CSF injection using heparin as anticoagulant (13 units ofsodium heparin/mL blood). Four different assays were performed on theblood to determine the extent of HSC mobilization. Some of these assaysare surrogate measures of mobilization that are commonly used in theclinical setting; that is they measure associated changes, not HSCcontent directly. These are total blood leukocyte counts and numberscolony forming cells (CFC) in the blood. The third assay based onphenotypic analysis (expression of a panel of surface markers whichdefine long-term reconstituting HSC), while directly measuring HSC, alsodetect some progenitor cell populations. Phenotypic enumeration iscommonly used to determine HSC mobilization in human patients (adifferent array of cell-surface markers are used in mice). However theonly assay that will truly determine the level of HSC mobilization intothe blood remains transplantation of mobilized blood into lethallyirradiated recipients, and following reconstitution, analyse whetherthese transplanted blood cells have indeed contributed to the bonemarrow and blood reconstitution in the lethally irradiated host.

These four assays were performed on the mobilized blood collected fromthese mice as follows:

A) Blood leukocyte counts were measured on a Sysmex KX21 automatedhaematology analyser (Roche).

B) Colony forming cell assay. A portion of the blood was subject to redcell lysis (blood was incubated in 150 mM NH₄CL, 10 mM NaCO₂, 1 mM EDTAfor 10 minutes on ice, then red cell remnants removed by washing twicein PBS with 2% newborn calf serum). Remaining nucleated blood leukocytes(exactly 50.000) were then added to 1 mL colony assay counting media induplicate, and incubated at 37° C. in 5% CO₂ to allow cell division intoclonogenic colonies. Following 7 days incubation, the number ofleukocyte colonies were enumerated manually using an invertedmicroscope. Each colony (defined as >50 cells) is derived from a singlehaematopoietic progenitor cell. The number of colony forming cells (CFC)per mL of blood can then been calculated. The colony assay countingmedia contains: Iscove's modified Dulbeccos medium (1×IMDM), 35% foetalcalf serum, Penicillin 125 μg/mL, Genamycin 16 μg/mL, L-glutamine 2 mM,recombinant mouse Interleukin 3 at 10 ng/mL, recombinant mouseInterleukin 6 at 50 ng/mL, recombinant mouse Stem cell factor at 50ng/mL.

C. Phenotypic HSC analysis. A portion of lysed blood (red blood celllysis method above) was stained using the following panel of directlyconjugated monoclonal antibodies at a concentration of 2.5 μg/10⁸cells/mL; a) biotinylated antibodies against lineage-specific antigensCD3, CD5, B220/CD45R, CD11b, Gr-1 or Ly-6C/G, CD41, Ter119 together withstreptavidin conjugated to Pacific blue; b) fluorescein isothiocyanate(FITC) conjugated anti-CD48; c) phycoerythrin (PE) conjugatedanti-SLAM/CD150; d) allophycocyanin (APC) conjugated anti-CD117/KIT ande) PECy7 conjugated anti-Sca-1. HSC are cells with the phenotypeLineage-negative, Sca-1-positive, CD117-positive, CD48-negative andCD150-positive¹¹.

D. Competitive Long-term reconstitution assay following transplant ofmobilized blood. These experiments use two strains of mice congenic forCD45 isoforms: The C57BL/6 strain which expresses CD45.2 isoform on allhematopoietic cells, and the B6.SJL strain which expresses the CD45.1isoform on all hematopoietic cells. This enables donor test cells to bedistinguished from host cells and competitive cells based on theexpression of either CD45.1 or CD45.2 antigens. Recipient mice for thetransplant (8-week old congenic B6.SJL females CD45.1⁺, 6 mice/group)received a lethal dose of irradiation on the day before the experiment(total 11.0 Gy given as two doses of 5.5 Gy, 4 hours apart—to minimizegut toxicity). Next day, each irradiated mouse was transplanted with 25μL of whole heparinized mobilized blood (pooled from either wild-type orE-selectin knockout donors, both are in the C57BL/6 background withCD45.2⁺ phenotype), mixed together with 200,000 competing healthy bonemarrow cells from the congenic B6.SJL strain (CD45.1⁺). Cells areinjected via the retro-orbital route, engraft and reconstitute thelethally-irradiated bone marrow and promote survival of the recipientmice. At 16 weeks post transplant, blood is collected from the recipientmice. Blood leukocytes will either be CD45.2⁺ (hence derived from themobilized blood) or CD45.1⁺ (derived from the transplanted healthycompeting bone marrow). To determine the phenotype of the blood,following red cell lysis, blood is stained with the following antibodiesfor phenotypic analysis: CD45.1-PE, CD45.2-FITC, CD11b-ape, B220-peCY7.The percentage of CD45.2⁺ blood reconstitution in recipient mice isdirectly proportional to the number of CD45.2⁺ HSC that were initiallypresent in the volume of mobilized blood transplanted. For ease ofinterpretation, these data have been converted into the number of‘reconstitution units’ (RU) measured per mL of blood¹². Onereconstitution unit is arbitrarily defined as the constant number of HSCpresent in 100.000 BM cells from healthy untreated mice. The number ofRU in mobilized donor blood is calculated as follows: donor RU=% donorcells×C/(100−% donor cells) where C is the number of competing RU giventogether with the mobilized blood^(12,13). For example, if 25 μL ofmobilized blood from a CD45.2⁺ mouse injected in competition with200.000 healthy CD45.1⁺ bone marrow cells (2 competing RU) into alethally-irradiated recipient resulted in a 50:50 reconstitution ofCD45.2⁺ and CD45.1⁺ blood cells, then the number of reconstitution unitsin the original mobilized blood was 2. This method is standard forcalculating the true HSC content in a donor sample. The number of actuallong-term reconstituting HSC within 100,000 healthy bone marrow cells (1RU) is between 2-3 HSC.

Repeated Rounds of Cyclophosphamide Chemotherapy

Wild-type and E-selectin KO mice were first administered 8 consecutiverounds of chemotherapy. The chemotherapeutic agent used was thealkylating agent cyclophosphamide administered as a singleintraperitoneal injection at 200 mg/kg. Mice were given 8 rounds ofcyclophosphamide chemotherapy at fortnightly intervals, then left torecover for 6 weeks before administration of G-CSF (total 1 mg/kg asabove) and blood collected 3 days later at HSC mobilization. Blood wasanalyzed for total leukocyte count, Colony forming cells and phenotypicHSC as described above.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

BIBLIOGRAPHY

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1.-47. (canceled)
 48. A composition comprising an E-selectin antagonist,wherein the E-selectin antagonist is not naturally occurring, and atleast one mobilizer of hematopoietic stem cells or progenitor cells. 49.The composition of claim 48, wherein the E-selectin antagonist isselected from the group consisting of an antigen binding molecule thatis immuno-interactive with E-selectin, a peptide that binds toE-selectin and that blocks cell-cell adhesion, a carbohydrate mimetic ofan E-selectin ligand, and a peptide mimetic of an E-selectin ligand. 50.The composition of claim 48, wherein the E-selectin antagonist is acarbohydrate mimetic of an E-selectin ligand.
 51. The composition ofclaim 48, wherein the at least one mobilizer is selected from the groupconsisting of: AMD3100, interleukin-1 (IL-I), interleukin-3 (IL-3),interleukin-6 (IL-6), interleukin-11 (IL-11), interleukin-7 (IL-7),interleukin-8 (IL-8), interleukin-12 (IL 12), granulocyte colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), macrophage colony stimulating factor (M-CSF), stem cellfactor, FLT-3 ligand, matrix metalloproteinase 2 (MMP2), MMP9, cathepsinG, elastase, cathepsin K, dipeptidyl peptidase-1, Mip-1α, Groβ, a smallinterfering RNA (siRNA) molecule specific for CXCL12, an antisensemolecule specific for CXCL12, Fucoidan, and sulfated dextran.
 52. Thecomposition of claim 48, wherein the at least one mobilizer is G-CSF.53. The composition of claim 48, wherein the E-selectin antagonist is acarbohydrate mimetic of an E-selectin ligand and the at least onemobilizer is G-CSF.
 54. The composition of claim 48, further comprisingan agent that targets rapidly dividing cells and/or disrupts the cellcycle or cell division.
 55. The composition of claim 54, wherein theagent is a chemotherapeutic agent or a radiosensitizing agent.
 56. Thecomposition of claim 49, further comprising an agent that targetsrapidly dividing cells and/or disrupts the cell cycle or cell division.57. The composition of claim 56, wherein the agent is a chemotherapeuticagent or a radiosensitizing agent.
 58. The composition of claim 50,further comprising an agent that targets rapidly dividing cells and/ordisrupts the cell cycle or cell division.
 59. The composition of claim58, wherein the agent is a chemotherapeutic agent or a radiosensitizingagent.
 60. The composition of claim 51, further comprising an agent thattargets rapidly dividing cells and/or disrupts the cell cycle or celldivision.
 61. The composition of claim 60, wherein the agent is achemotherapeutic agent or a radiosensitizing agent.
 62. The compositionof claim 48, further comprising a medicament for treating animmunocompromised condition.
 63. The composition of claim 62, whereinthe medicament is selected from the group consisting of an anemiamedicament, a thrombocytopenia medicament, a neutropenia medicament, andan anti-infective agent.
 64. The composition of claim 49, furthercomprising a medicament for treating an immunocompromised condition. 65.The composition of claim 64, wherein the medicament is selected from thegroup consisting of an anemia medicament, a thrombocytopenia medicament,a neutropenia medicament, and an anti-infective agent.
 66. Thecomposition of claim 50, further comprising a medicament for treating animmunocompromised condition.
 67. The composition of claim 66, whereinthe medicament is selected from the group consisting of an anemiamedicament, a thrombocytopenia medicament, a neutropenia medicament, andan anti-infective agent.
 68. The composition of claim 51, furthercomprising a medicament for treating an immunocompromised condition. 69.The composition of claim 68, wherein the medicament is selected from thegroup consisting of an anemia medicament, a thrombocytopenia medicament,a neutropenia medicament, and an anti-infective agent.
 70. Thecomposition of claim 48, further comprising a pharmaceuticallyacceptable carrier.